Chapter 12

Protein Quantitation and Analysis of Purity

Eva M. Campion, Sinéad T. Loughran, and Dermot Walls

Abstract
The accurate quantitation of proteins and an analysis of their purity are essential in numerous areas of scientific
research, and is a critical factor in many clinical applications. The large number and variety of techniques
employed for this purpose is therefore not surprising. The selection of a suitable assay is dependent on such
factors as the level of sensitivity required, the presence of interfering agents, and the composition of the pro-
tein itself. Here, protocols for the most commonly used protein determination methodologies are outlined,
as well as for the more recently adapted technique of quantitative immuno-Polymerase Chain Reaction.

Key words Protein, Lowry, Bradford, BCA, ELISA, Quantitative immuno-PCR (qIPCR)

1 Introduction

The ability to easily and accurately quantitate total protein content

in a given sample is a fundamental requirement of many biological
studies. Indeed, the routine measurement of total protein content
is a well-established essential step in many areas of basic biochemi-
cal research and routine clinical practice [1].
Numerous and varied methods to assay total protein content
have been described in the literature. The most commonly uti-
lized methods rely on (1) the intrinsic ability of protein mole-
cules to absorb Ultraviolet (UV) light (UV absorption) [2], (2)
the use of protein-binding dyes exploiting either colorimetric or
fluorescent-based detection (Bradford Assay, Silver staining,
NanoOrange™) [3–5], and (3) the reduction of copper in the
presence of a chromogenic reagent [Lowry and Bicinchoninic
acid (BCA) assays] [6–8]. Since each of these methods has its
own strengths and weaknesses (see Note 1) [9], none of these
assays should be employed without first considering its suitabil-
ity for the application in question (see Table 1). Even fluorescent
assays developed in recent years to alleviate difficulties experi-
enced with absorbance based assays (for example, OPA (o-phthal-
dialdehyde), fluorescamine, and NanoOrange™) are not without

Dermot Walls and Sinéad T. Loughran (eds.), Protein Chromatography: Methods and Protocols, Methods in Molecular Biology,
vol. 1485, DOI 10.1007/978-1-4939-6412-3_12, © Springer Science+Business Media New York 2017

225
226 Eva M. Campion et al.

Table 1
A comparison of UV absorption, Bradford, Lowry, BCA, and fluorescent protein assay methods

Method name Advantages Disadvantages

UV absorption • Simple, fast • Many buffer components absorb strongly in this
• Inexpensive region
• Sample is recoverable • The presence of nucleic acid can greatly influence
the absorption
• Least sensitive method
Bradford Assay • Simple, fast • Nonlinear standard curve over wide ranges
• Inexpensive • Response to different proteins can vary widely:
• Very sensitive choice of standard is very important
• Compatible with a wide
range of buffers
Lowry Assay • Sensitive • Time consuming
• Commonly referenced • Susceptible to many interfering compounds
procedure • Variation in the content of tyrosine and
• Easily adapted to tryptophan residues will influence the assay
microplate format
BCA Assay • Very sensitive • The reaction does not go to completion when
• Rapid performed at room temperature or 37 °C
• Compatible with a wide (difficult when prepping large numbers of
range of buffers samples)
• Little variation in response
between different proteins
NanoOrange™ • Highly sensitive • Compatible with reducing agents not detergents
• Little variation in response
between different proteins
• Simple, fast
Fluorescamine • Simple, fast • Sensitivity depends on number of amines present
• Very Sensitive in sample
• Poor water solubility
• Not compatible with amine-containing buffers
OPA • Very sensitive • Sensitivity depends on number of amines present
• Simple, fast in sample
• Water soluble • Not compatible with amine-containing buffers
• Inexpensive
CBQCA • Highly sensitive • Sensitivity depends on number of amines present
• Linear over an extended in sample
range of protein • Not compatible with buffers containing amines
concentration or thiols
• Compatible with
detergents and lipophilic
proteins
 Protein Quantitation and Analysis of Purity 227

their weaknesses (see Table 1) and the Bradford and Coomassie

assays remain the most widely referenced in the literature [8].
There is in fact no absolute method that produces accurate
results in every instance and often it is necessary to employ more
than one type of protein assay [10, 11]. In addition to total pro-
tein concentration, the specific activity of a particular target pro-
tein in a sample is of significance when proteins are being purified
or when different protein samples are being compared [4]. In
broad terms, measurement of the level of a specific protein of
interest may be undertaken by one of two methods, a specific
biological assay (or bioassay), or an immunoassay. The specificity
of an immunoassay relies on the interaction between the protein
and an antibody, and determining the quantity of bound anti-
body in an immunoassay is routinely achieved by virtue of either
using a labeled primary antibody, or detecting the latter using a
secondary antibody that is itself labeled [12]. There are many
immunoassay formats possible and labels that are used include
enzymes [e.g., enzyme linked-immunosorbent assay (ELISA)],
radioactive labels [radioimmunoassay (RIA)], magnetic labels
[e.g., magnetic immunoassay (MIA)], fluorescent tags [as used
in flow cytometry/fluorescence activated cell sorting (FACS)
analysis and fluorescence microscopy], or a piece of DNA [as in
real-time quantitative immuno-Polymerase Chain Reaction
(qIPCR)]. More recently, protein mass spectrometry has become
an emerging method for protein quantitation and is discussed
elsewhere in this volume.
A pure protein is one that is free from quantifiable levels of
impurities [13]. Any purity determination is only as reliable as
the analytical methods used, and factors such as the structural
properties of the protein itself, the amount of protein available,
the nature of potential contaminants in the sample, and the
accuracy of the estimate required, should always be considered
when selecting the method of analysis [14]. In reality, it may
only be necessary to ensure the sample is free of contaminating
products that may affect the application in question, and thus
aspects of the process such as the intended use (e.g., bulk
enzyme preparations, protein crystallography, primary sequence
analysis, or therapeutic applications), the source of the protein
(animal tissue, human serum, recombinant microorganisms, or
hybridomas) and the purification processes employed should all
accordingly dictate the extent of analysis required [4]. Here,
general protocols for the following most commonly employed
methods of protein estimation and purity are described:
Ultraviolet (UV) Protein Absorption Assays, the Bradford and
Lowry assays, Macro- and Micro-Bicinchoninic acid (BCA)
assays, ELISA, Sodium Dodecyl Sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and associated staining methods,
Western Immunoblotting, and several formats for qIPCR.
228 Eva M. Campion et al.

2 Materials

2.1 Protein 1. Cuvettes (Quartz for wavelengths <215 nm) (Sarstedt).

Determination 2. Buffer solution in which the protein sample is dissolved (for
by Ultraviolet (UV) blanking, see Note 2).
Light Absorption
3. UV Spectrophotometer.

2.2 Preparation 1. Stock solution: prepare a stock solution of a standard protein

of a Standard Curve e.g., bovine serum albumin (BSA), lysozyme, albumin, or
γ-globulin (see Note 3) at a suitable concentration (1–5 mg/
mL see Note 4), and in the same buffer as the protein of
unknown concentration.
2. Buffer (for blanking).

2.3 UV Protein 1. Distilled H2O (dH2O).

Absorption Assay 2. Lab-wipes (Thermo-Scientific).
Using Microvolume
3. Buffer for blanking (see Note 5).
Spectroscopy
4. Nanodrop® 2000 Spectrophotometer (Thermo-Scientific).

2.4 The Bradford 1. Protein standard solutions (e.g., 1 mg/mL BSA): dilute in the
Protein Assay range 20–100 μg/mL in a total volume of 100 μL. Aliquot
and store at −20 °C.
2. Buffer (for blanking, see Note 6).
3. 1 M sodium hydroxide (NaOH) (see Note 7).
4. Bradford reagent available from Sigma-Aldrich, or prepare
oneself by adding 100 mg Coomassie brilliant blue G-250
(Thermo-Scientific) to 50 mL 95 % (v/v) ethanol. When
Coomassie brilliant blue G-250 has dissolved add 100 mL 85 %
(w/v) phosphoric acid and stir overnight. Dilute to 1 L with
dH2O. Store for up to 3 months at 4 °C (see Note 8).
5. Cuvettes (see Note 9) (Sarstedt).

2.5 The Lowry 1. Lowry solution A: 2 % (w/v) sodium carbonate (Na2CO3) in

Protein Assay 0.1 M sodium hydroxide (NaOH). Store for up to 3 months at
room temperature.
2. Lowry solution B: 1 % (w/v) copper sulfate pentahydrate
(CuSO4·5H2O, Sigma-Aldrich) in dH2O. Store for up to 1
year at room temperature.
3. Lowry solution C: 2 % (w/v) sodium potassium tartrate
(NaKC4H4O6·4H2O) in dH2O. Store for up to 3 months at
room temperature.
4. Lowry working solution: Prepare immediately before use;
Lowry Solution A: Lowry solution B: Lowry Solution C in the
ratio 100:1:1 (v:v:v), respectively.
 Protein Quantitation and Analysis of Purity 229

5. Folin–Ciocalteu Reagent: Available as 2 N reagent (Sigma-

Aldrich). Dilute 1:1 in dH2O. This solution is light sensitive
and should be prepared just prior to use and kept in a light-
protected container.
6. Protein standards: Prepare a dilution series of standard pro-
tein e.g., albumin in the range 0–100 μg/mL in a total vol-
ume of 1 mL.
7. Buffer for blanking (see Note 10).

2.6 The 1. Macro-BCA Reagent A: Dissolve 1 g sodium bicinchoninate

Bicinchoninic Acid (BCA, Pierce), 2 g sodium carbonate (Sigma-Aldrich), 0.16 g
(BCA) Assay sodium tartrate (Sigma-Aldrich), 0.4 g NaOH, and 0.95 g
sodium bicarbonate in dH2O. Adjust the pH to 11.25 with
2.6.1 Macro-BCA Assay
10 M NaOH and bring to 100 mL with dH2O. Stable for 1
year at room temperature.
2. Macro-BCA Reagent B: Dissolve 0.4 g CuSO4·5H2O in 10 mL
distilled water. Stable for 1 year at room temperature.
3. Macro-BCA Working solution: Mix 50 volumes of Macro-BCA
reagent A with 1 volume of Macro-BCA reagent B (Prepare fresh
before use). The working solution should be green in color.
4. Glass or disposable polystyrene cuvettes (Sarstedt).
5. Buffer for blanking (see Note 11).

2.6.2 Micro-BCA Assay The Micro-BCA assay uses three reagents whose concentrations
are significantly higher than the two reagents used in the Macro-
BCA format. Here, BCA is prepared as a separate reagent in order
to avoid its precipitation.
1. Micro-BCA assay reagent A: Dissolve 8 g sodium carbonate
monohydrate, 1.6 g sodium tartrate in dH2O, adjust the pH to
11.25 with 10 M NaOH, and bring to 100 mL with
dH2O. Stable for 1 year at room temperature.
2. Micro-BCA assay reagent B: Dissolve 4 g BCA in 100 mL
dH2O. Stable for 1 year at room temperature.
3. Micro-BCA assay reagent C: Dissolve 0.4 g CuSO4·5H2O in
10 mL dH2O. Stable for 1 year at room temperature.
4. Micro-BCA assay solution (prepare fresh): 25:25:1 (v/v/v)
Micro-BCA assay reagent A/Micro-BCA assay reagent B/
Micro-BCA assay reagent C.
5. Buffer for blanking (see Note 11).

2.7 Immunoassay: 1. Microtiter plates (Biosciences).

Indirect ELISA 2. Antigen-coating buffer: 0.1 M Na2CO3/sodium bicarbonate
(NaHCO3) pH 9.6 (adjust the pH if necessary using HCl).
3. Phosphate buffered saline (PBS).
230 Eva M. Campion et al.

4. Assay buffer: 20 mM PBS pH 7.4 (adjust pH to 7.4 by adding

dilute HCl or NaOH if necessary), 0.05 % (v/v) Tween 20.
5. Blocking buffer: 20 mM PBS, 5 % (w/v) nonfat dried milk (or
other suitable blocking agent see Note 12).
6. Primary antibody (diluted to the optimal concentration in
blocking buffer immediately before use. This concentration
should be experimentally determined, for most applications
dilution in the range 1:200 to 1:1000 is sufficient, consult
manufacturer’s guidelines).
7. Conjugated secondary antibodies directed toward the primary
antibody (diluted to the optimal concentration in blocking
buffer immediately before use).
8. Substrate e.g., for peroxidase system: add 100 μL of 0.8 mg/
mL o-phenylenediamine dihydrochloride (OPD; Sigma-
Aldrich) dissolved in 0.1 M phosphate-citrate buffer pH 5.0,
containing 0.4 mg/mL urea hydrogen peroxide (UHP; Sigma-
Aldrich) and incubate at room temperature for 30 min.
9. Stop solution: alkaline phosphatase—3 M NaOH;
Peroxidase—3 M HCl or 3 M H2SO4.

2.8 Sodium Dodecyl 1. Leupeptin (Sigma-Aldrich): Dissolve 2 mg/mL leupeptin in

Sulfate- dH2O and store at −20 °C.
Polyacrylamide Gel 2. Aprotinin (Sigma-Aldrich): Make 0.1 M stock solution of
Electrophoresis aprotinin in dH2O and store at −20 °C.
(SDS-PAGE) 3. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich):
of Proteins Make 100 mg/mL PMSF in isopropanol and store at −20 °C
in the dark.
4. Suspension buffer: 0.1 M NaCl, 0.01 M Tris–HCl (pH 7.6),
0.001 M NaOH-EDTA (pH 8.0), 1 μg/mL leupeptin, 1 μg/
mL aprotinin, 100 μg/mL PMSF. Store at 4 °C.
5. 2× SDS loading buffer (Sample Buffer): 100 mM Tris–HCl
(pH 7.6), 4 % (w/v) SDS, 20 % (v/v) glycerol, 10 % (v/v)
2-mercaptoethanol, 0.2 % (w/v) bromophenol blue. Store at
room temperature.
6. Acrylagel (National Diagnostics). Acrylagel is toxic, and a
known carcinogen. Consult the corresponding material safety
data sheet (MSDS) before use.
7. Bis-Acrylagel (National Diagnostics). Bis-acrylagel is an irri-
tant. Consult the corresponding MSDS before use.
8. 1.5 M Tris–HCl (pH 8.8).
9. 1 M Tris–HCl (pH 6.8).
10. dH2O.
11. 10 % (w/v) sodium dodecyl sulfate (SDS) in dH2O.
 Protein Quantitation and Analysis of Purity 231

12. 10 % (w/v) ammonium persulfate (APS) dH2O (prepare

freshly) (Sigma-Aldrich). APS is a strong and harmful oxidiz-
ing agent. Consult the corresponding MSDS before use.
13. TEMED (N,N,N′,N′-Tetramethylethylenediamine).
14. 5× Tris–glycine running buffer (Electrode Buffer): 15.1 g Tris
base, 95.4 g glycine, 50 mL 10 % (w/v) SDS. Make up to 1 L
with dH2O and store at room temperature.
15. 1× Tris–glycine running buffer: 200 mL 5× Tris–glycine run-
ning buffer, 800 mL dH2O. Store at room temperature.
16. Molecular weight marker (prestained) (GE Healthcare).
17. ATTO protein gel electrophoresis system.

2.9 Staining of 1. Coomassie brilliant blue G-250 solution (Sigma-Aldrich).

SDS-PAGE Gels 2. Destain: 450 mL methanol, 450 mL dH2O, 100 mL glacial
2.9.1 Coomassie Brilliant acetic acid. Store at room temperature.
Blue Staining of SDS-PAGE
Gels 1. Fixing Solution: methanol, acetic acid, and formalin
(40:10:0.05 by volume).
2.9.2 Silver Staining 2. Wash Solution: ethanol, acetic acid, and water (10:5:85, by
of SDS-PAGE Gels volume).
3. Silver nitrate solution (Thermo-Scientific): or 0.2 % (w/v)
AgNO3, 0.076 % formalin (prepare fresh). Silver Nitrate is
harmful. Consult the corresponding Material Safety Data
Sheet (MSDS) before use.
4. Oxidizing solution: 3.4 mM potassium dichromate and
3.2 mM nitric acid.
5. Developing solution: 0.28 mM sodium carbonate and 1.9 %
(v/v) formaldehyde. Formaldehyde is toxic, consult the cor-
responding MSDS prior to use.
6. Stop solution: methanol, acetic acid (50:12).
7. 1 % (v/v) acetic acid.

2.10 Western 1. Bio-Rad Trans-Blot® SD semi-dry electrophoretic transfer cell.

Blotting 2. Transfer buffer: 750 mL dH2O, 2.9 g glycine, 5.8 g Tris base,
3.7 mL 10 % (w/v) SDS, 200 mL methanol. Adjust volume
to 1 L with dH2O and store at 4 °C (Storing at this tempera-
ture is critical).
3. 1× Tris buffered saline (TBS): 6.1 g Tris base, 8.8 g NaCl,
800 mL dH2O. Adjust the pH to 7.5 with HCl and adjust the
volume to 1 L with dH2O. Store at room temperature.
4. TBS-T: 1 L 1× TBS, 1 mL Tween 20 (Sigma-Aldrich). Store at
room temperature.
5. Blocking buffer: 5 g non fat dry milk powder (or other appropri-
ate blocking agent see Note 12), 100 mL TBS-T. Store at 4 °C.
232 Eva M. Campion et al.

6. Nitrocellulose blotting membrane (Labkem).

7. 3MM filter paper (Whatman).
8. Scalpel blade (Swann-Morton).
9. Ponceau S (Sigma-Aldrich).
10. dH2O.
11. Primary antibody (diluted to optimal working concentration
in blocking buffer immediately before use. This concentration
should be experimentally determined, for most applications
dilution in the range 1:200 to 1:1000 is sufficient; consult
manufacturers’ guidelines).
12. Conjugated secondary antibody directed toward the primary
antibody (diluted to an optimal working concentration in
blocking buffer immediately before use).
13. Substrate (e.g., 5-Bromo-4-chloro-3-indolyl phosphate/Nitro
Blue Tetrazolium (BCIP/NBT, Sigma-Aldrich), or
3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich).

2.11 qIPCR 1. Template DNA (e.g., linearized pUC19 plasmid DNA).

2.11.1 Preparation 2. Template-specific oligonucleotide primers: 5′-biotinylated for-
of Biotinylated DNA Label ward primer and unmodified reverse primer.
3. PCR purification kit e.g., QIAquick PCR purification kit
(Qiagen).
4. Agarose powder (Sigma-Aldrich).
5. 50× Tris-acetate/EDTA electrophoresis buffer (TAE): 242 g
Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 M NaOH-
EDTA (pH 8.0). Adjust to 1 L with dH2O and store at room
temperature.
6. 1× TAE buffer: 20 mL 50× TAE, 980 mL dH2O. Store at
room temperature.
7. Loading dye: 40 % (w/v) sucrose, 0.25 % (w/v) bromophenol
blue. Store at room temperature.
8. SYBR Safe DNA gel stain (Invitrogen).
9. Horizontal agarose gel electrophoresis system
(Sigma-Aldrich).
10. DNA size markers (e.g., 100 bp ladder from Invitrogen).
11. PCR reaction mixture: 5 μL 10× PCR buffer, 1 μL of each
dNTP (200 μM), 1 μL of each primer (100 μmol/L), 1.25 U
Taq polymerase, 50 ng template DNA, bring to 50 μL with
molecular grade H2O.
12. Prepare a 1.5 % (w/v) agarose gel: Dissolve the agarose in 1×
TAE by boiling, with intermittent mixing until completely dis-
solved. Add SYBR Safe DNA gel stain according to the manu-
facturer’s instructions. Cast the gel according to the instructions
accompanying the apparatus being used.
 Protein Quantitation and Analysis of Purity 233

13. G-50 sephadex column (Roche).

14. Cuvettes (Sarstedt).

2.11.2 Preparation 1. Detection antibody (in PBS, 1 mM EDTA, pH 8.0 (adjust the
of Antibody/DNA Label pH of buffer by adding dilute HCl/NaOH if necessary).
Conjugate 2. 2-iminothiolane (Traut’s reagent, Sigma-Aldrich).
3. 1 M glycine-NaOH pH 7.3.
4. 5′ amino-modified DNA (in PBS, 1 mM EDTA, pH 7.2,
adjust the pH of the buffer by adding dilute HCl or NaOH if
necessary).
5. Sulfo-succinimidyl4-[ N- maleimidomethyl]cyclohexane-1-
carboxylate (SMCC).
6. Zeba 2 mL desalt spin column (Thermo-Scientific).
7. Ion exchange column (Resource Q 1 mL, Biosciences).
8. 10 mM Tris–HCl pH 8.0.
9. 1.5 M NaCl.
10. Centricon YM-100 (Millipore).

2.11.3 Biotinylation 1. Detection antibody (in amine-free buffer, pH 7.2–8.0).

of Detection Antibody 2. Biotinamido-caproate-N-hydroxysuccinimide ester (BNHS)
1 mg/mL: Dissolve 1 mg of BNHS in 1 mL dimethyl sulfox-
ide (DMSO) immediately before use.
3. 1 M NaHCO3 pH 8.5 (adjust the pH if necessary using NaOH
or H2SO4).
4. PD-10 gel filtration column (Sephadex G-25 M), GE
Healthcare or dialysis tubing (Sigma-Aldrich).
5. PBS pH 7.4 (adjust pH to 7.4 by adding dilute HCl or NaOH
if necessary).

2.11.4 qIPCR Assay 1. Wash buffer: 0.154 M NaCl, 5 mM Tris–HCl pH 7.75, 0.02 %
(Common Components) (w/v) sodium azide (NaN3) (see Note 13).
2. Blocking buffer: PBS, 0.1–1 % (w/v) BSA (or other suitable
blocking agent, see Note 14) and 0.05 % (v/v) Tween 20.
3. Antigen/unknown samples: dilute the antigen standard, pre-
paring a suitable concentration range (see Note 15).
4. Ultrapure water (see Note 16).

2.11.5 Assay Format 1. Capture antibody (diluted to optimal working concentration

I (Additional Components) in 0.2 M NaH2PO4; see Note 17).
2. Polypropylene PCR plate (see Note 18).
3. Biotinylated detection antibody (diluted to optimal working
concentration in blocking buffer).
234 Eva M. Campion et al.

4. Streptavidin (5 nM in blocking buffer).

5. Biotinylated DNA label (0.7 pM in blocking buffer) (see
Subheading 3.11.1).

2.11.6 Assay Format II 1. Capture antibody (diluted at optimal concentration in 0.2 M

(Additional Components) NaH2PO4).
2. Polypropylene PCR plate (see Note 18).
3. DNA-conjugated detection antibody (see Subheading 3.11.2)
(diluted to optimal working concentration).

2.11.7 Assay Format III 1. Streptavidin-coated microtiter plates (Roche).

(Additional Components) 2. DNA-conjugated detection antibody (see Subheading 3.11.2)
(diluted to optimal working concentration).
3. Biotinylated capture antibody (diluted to optimal working
concentration).

2.11.8 Assay Format IV 1. Polypropylene PCR plate (see Note 18).

(Additional Components) 2. DNA-conjugated detection antibody (see Subheading 3.11.2)
(diluted at optimal concentration).
3. Antigen-coating buffer: 0.1 M Na2CO3-NaHCO3 pH 9.6
(adjust the pH if necessary using HCl).

2.11.9 Real Time PCR Real Time PCR mixture (assemble on ice):
(qPCR) Assay
1. 2 μL 10× PCR buffer (4 mM MgCl2) (Promega).
2. 200 μM dNTPs (dUTP, dGTP, dCTP, dATP) (Invitrogen).
3. Hot start Taq polymerase (Promega).
4. 200–400 nM primers [specific for DNA label (see
Subheading 3.11.1)] (Eurofins MWG operon).
5. 0.5 μL 50× SYBR green solution (vortex for 30 s before use)
(Quantace).
6. Uracil-N-glycosylase (UNG) (Bioline) (see Note 19).
7. Bring to 20 μL with molecular grade H2O (Sigma-Aldrich).
8. Filter/barrier tips (Anachem).

3 Methods

3.1 Protein Simple and rapid estimations of protein concentration can be made
Determination by UV by monitoring the absorbance of ultraviolet light. Absorbance of
Light Absorption near-UV light at 280 nm depends largely on the presence of aro-
matic amino acids, in particular tryptophan and tyrosine, and to a
much more minor extent on phenylalanine and disulfide bonds.
Absorption is affected by pH and ionic strength. In addition,
strong interference from nucleic acids is a particular problem at
 Protein Quantitation and Analysis of Purity 235

this wavelength. As a method for determining protein concentra-

tion, UV absorption gives no more than a quick and rough esti-
mate unless the protein preparation is pure and its extinction
coefficient is known. The sample is recoverable however, and the
user should proceed to one of the other assays below when a more
accurate determination of protein concentration is required.
1. Switch on the UV spectrophotometer and set the wavelength
at 280 nm. Leave the instrument to stabilize for 15–20 min.
2. Calibrate the instrument to zero absorbance using a water
blank. Ensure to use suitable cuvettes (quartz or other cuvettes
known to be transparent at the given wavelength).
3. Measure the A280 of the buffer used to prepare the sample. The
cuvette should be filled with a sufficient volume to cover the
aperture through which the light beam passes (do not allow
any bubbles to inhibit the path of the light).
4. Measure the absorbance of the protein sample by replacing the
buffer blank with a cuvette containing the protein sample.
5. If the A280 exceeds 2, dilute the sample using buffer and read
the absorbance again.

3.2 Preparation 1. Prepare a stock solution of a protein standard (e.g., BSA

of a Standard Curve 0.1 mg/mL) and set up dilutions for the preparation of a stan-
dard curve (see Table 2). The concentration of protein in the
samples must fall within the linear range of the standard curve.
2. Pipette duplicate aliquots of protein standards into microfuge
tubes and dilute appropriately (see Note 20) with a suitable
buffer, e.g., 0.15 M NaCl.
3. Mix well by inversion or using a vortex.
4. Carry out the assay as per Subheading 3.1.

Table 2
Preparation of protein standards for standard curve generation

Final concentration (e.g., BSA µg/ Volume of buffer

mL) Volume of stock µL (BSA 0.1 mg/mL) (µL)
0 0 100
10 10 90
20 20 80
30 30 70
40 40 60
50 50 50
236 Eva M. Campion et al.

5. Prepare a standard curve by plotting absorbance versus protein

concentration (see Note 21).
6. Use the equation of the line to calculate the protein concentra-
tions of the unknown samples.

3.3 UV Protein Microvolume spectrophotometers measure protein sample volumes

Absorption Assay in the range of 0.5–2 μL. These instruments allow fast, precise quan-
Using Microvolume tification of protein, while also preserving precious sample. There
Spectroscopy are numerous microvolume spectrophotometers on the market—all
with similar modus operandi. Generally, microvolume spectropho-
tometers employ fiber optic technology and use the inherent surface
tension of the liquids being analyzed to create a column between the
ends of the optical fibers (the sensors). In this way, the measurement
optical path is formed. Very small protein sample volumes (0.5–2 μL)
can therefore be rapidly analyzed spectrophotometrically without
the need for cuvettes or capillaries. The procedure for use with the
Nanodrop® 2000 instrument is outlined below.
1. Raise the sampling arm and clean the upper and lower sensor
surfaces with dH2O water and a lab-wipe to eliminate any
dried/leftover sample that might be present (cleaning in this
manner is sufficient to prevent any sample carryover).
2. Open the NanoDrop® software program and select the appro-
priate component (see Note 22).
3. Pipette 0.5–2 μL (see Note 23) of dH2O onto the sensor.
Carefully bring down the arm.
4. Follow the onscreen prompts to initialize/calibrate the
instrument.
5. Clean the sensors as before and pipette 0.5–2 μL (see Note 24) of
the buffer (for blanking) onto the sample surface. Lower the arm.
6. Follow the onscreen prompts to blank the instrument (see
Note 25).
7. Clean the upper and lower pedestal surfaces and pipette
0.5–2 μL of the sample onto the sensor and lower the arm.
8. Click “measure” and record the concentration of the sample,
now shown onscreen.
9. In order to analyze multiple samples, clean the sensor between
measurements. (Recalibration or reblanking is not necessary).
10. Once finished, clean the sensors and switch off the
instrument.

3.4 The Bradford The Bradford Assay is based on the formation of a complex between
Protein Assay the dye, Brilliant Blue G-250, and the proteins in a solution. The
absorption maximum of the dye shifts from 465 to 595 nm when
it complexes with protein and the amount of absorption observed
is proportional to the quantity of protein present. This is a simple,
 Protein Quantitation and Analysis of Purity 237

rapid, inexpensive assay, and unlike other protein assay procedures

such as Lowry and BCA, the Bradford assay is compatible with
reducing agents that are often used for the purposes of stabilizing
proteins in solution. The Bradford Assay is not suitable however, if
even low concentrations of detergents are present in the sample,
and in that case the BCA protein determination procedure is to be
recommended. Although several modified protocols exist [15,
16], the original method as described by Bradford is still the most
widely used formulation [3, 17] and is laid out here. The linear
concentration range of the Bradford assay when using BSA as stan-
dard is 0.1–1.5 mg/mL of protein.
1. Prepare the protein standards in the range 20–100 μg (see
Note 26) diluted in the same buffer as the unknown (e.g.,
H2O/0.15 M NaCl) in a final volume of 100 μL as per
Subheading 3.2 (see Note 27).
2. Prepare two blanks by pipetting 100 μL of buffer into two
microfuge tubes.
3. Pipette 20–100 μg of the protein sample of unknown concen-
tration into microfuge tubes in a total volume of 100 μL (if
desired, add an equal volume of 1 M NaOH to samples and
standards to prevent precipitation upon the addition of
Bradford reagent).
4. Add 5 mL Bradford reagent and mix well by inversion or using
a vortex. Allow the samples to stand for 2–60 min at room
temperature (avoid foaming as this will lead to poor
reproducibility).
5. Meanwhile, switch on the UV spectrophotometer and set the
wavelength at 595 nm. Leave the instrument to stabilize for
15–20 min.
6. Calibrate the instrument to zero absorbance using air as a
blank.
7. Transfer samples and standards to cuvettes and determine the
absorption at 595 nm.
8. Make a standard curve by plotting absorbance at 595 nm versus
protein concentration. Use the standard curve to determine the
concentration of protein in the unknown sample (see Note 28).

3.5 The Lowry The Lowry assay involves two reactions, the first resulting in the
Protein Assay formation of a copper ion complex with amide bonds, which forms
reduced copper in alkaline solution. The second reaction is the
reduction of the Folin–Ciocalteu reagent, mainly by the reduced
copper–amide bond complex, but also by tyrosine and tryptophan
residues. The reduced reagent is blue and is thus detectable using
a spectrophotometer, detectable in the region of 500–750 nm.
The assay has a dynamic range of 1–100 μg of protein. A major
238 Eva M. Campion et al.

limitation of the Lowry assay is the fact that it is sensitive to a

considerable range of agents that are frequently found in many
lysis buffers commonly used during cell lysis. In addition, this assay
is strongly biased to those proteins that are rich in tyrosine and
tryptophan. There are several commercial suppliers of the modified
Lowry assay (Roche, Pierce, Bio-Rad, Sigma, and Thermofisher)
which may be employed here. However, different formulations
often do not give equivalent results even when using the same
standards, dilution buffers, and in the presence of the same inter-
fering substances. For this reason, it is important to be consistent
with the choice of commercial assay [5].
1. Prepare the protein standards (see Note 29) in the range
0–100 μg (as per Subheading 3.2) in a total volume of 1 mL.
2. Pipette 200 μL of each standard and the samples of unknown
protein concentration into microfuge tubes. Prepare two blank
tubes using 200 μL water/buffer.
3. To 200 μL of sample, blank or standard, add 1 mL of freshly
prepared Lowry working solution. Let the solution stand at
room temperature for 10–30 min (see Note 30).
4. Add 100 μL of diluted Folin–Ciocalteu reagent, vortex imme-
diately (essential for obtaining reproducible results), and stand
for 30–60 min (do not exceed 60 min) at room temperature in
the dark (complete mixing of the reagent must be accom-
plished quickly to avoid decomposition of the reagent before it
reacts with protein) (see Note 31).
5. Transfer the samples to cuvettes.
6. Set the spectrophotometer to 660 nm and leave to stabilize for
15–20 min (see Note 32). Zero the instrument using the blank
sample. Measure the absorbance of all the samples in turn.
7. Plot a standard curve of absorbance as a function of initial pro-
tein concentration and use it to determine the unknown pro-
tein concentrations (see Note 33).

3.6 The The principle of the Bicinchoninic acid (BCA) assay is similar to
Bicinchoninic Acid that of the Lowry procedure, in that both rely on the formation of
(BCA) Assay a Cu2+-protein complex under alkaline conditions, followed by
reduction of the Cu2+ to Cu1+. Here, BCA reagent replaces the
Folin–Ciocalteu reagent, and the amount of reduction is propor-
tional to the protein present as before. BCA forms a blue–purple
complex with Cu1+ in alkaline solutions, the appearance of which
can be monitored by absorbance at 562 nm. Unlike the Lowry
assay, BCA does not interact with detergents and is less susceptible
to interference from other compounds that may be found in buf-
fers used for cell lysis and protein preparation. Some reducing or
chelating agents such as DTT and EGTA are best avoided however
as they interfere by either reducing or sequestering Cu2+.
 Protein Quantitation and Analysis of Purity 239

3.6.1 Macro-BCA Assay 1. Prepare the protein standards in the range 0–100 μg diluted in
dH2O (as per Subheading 3.2) in a final volume of
100 μL. Prepare a blank tube using 100 μL water/buffer.
2. Pipette 100 μL of sample/blank/standard into test tubes, add
2 mL of the working solution and mix thoroughly.
3. Incubate for 15 min at 60 °C (see Note 34).
4. Cool the samples to room temperature (see Note 35) (the
color is stable up to 1 h following incubation at 60 °C).
5. Mix the samples using a vortex and transfer to cuvettes.
6. Set the spectrophotometer to read A562 and leave to stabilize
for 15–20 min. Zero the instrument, blank, and measure the
absorbance of every sample in turn.
7. Plot a standard curve of absorbance as a function of initial pro-
tein concentration and use it to determine the protein concen-
trations in the samples (see Subheading 3.2).

3.6.2 Micro-BCA Assay 1. Set up microfuge tubes containing samples and known amounts
of standard protein in the range of 0.5–20 μg with a final sam-
ple volume of 500 μL. Prepare two blank tubes using 500 μL
water/buffer.
2. Add 500 μL of micro-BCA assay solution to each tube, vor-
tex, and incubate the sample for 15 min at 60 °C (see Notes
34 and 36).
3. Cool samples to room temperature (see Note 35).
4. Vortex the samples and read the absorbance at 562 nm as
described in Subheading 3.1.

3.7 Immunoassay: ELISAs are common antibody-based tests that are used to detect
Indirect ELISA antigens or other antibodies in a sample. It is imperative that a robust
antibody directed against the protein of interest (i.e., the antigen) be
available to the investigator for the purpose of target protein quanti-
tation. There are many direct and indirect formats available, and the
specific detection antibody can be covalently linked to a reporter
enzyme or can alternatively be itself detected using an enzyme-
labeled secondary antibody. Many ELISAs utilize chromogenic sub-
strates, though newer assays employ fluorogenic markers enabling
much higher sensitivities to be achieved. The amount of target pro-
tein in a sample is inferred from the level of activity/signal from the
reporter. Traditional ELISA can be performed in four different for-
mats; direct, indirect, sandwich, and competitive [18–21]. Here, the
format typical of an indirect ELISA is outlined.

3.7.1 Antigen Coating 1. Dilute the antigen standard and the unknown sample in the
to Microtiter Plates antigen-coating buffer to set up a concentration range of 0.1–
10 μg/mL.
240 Eva M. Campion et al.

2. Add 50 μL of these to separate wells of the microtiter plate. Set

up control wells containing no antigen and standard wells con-
taining serially diluted antigen to provide data to construct a cali-
bration curve. It is advisable to prepare triplicate wells in all cases.
3. Incubate the plates at room temperature for 5–6 h on a microti-
ter plate shaker, or alternatively overnight at 4 °C.

3.7.2 Blocking 1. Empty the wells and wash three times with assay buffer fol-
lowed by three further washes with PBS (see Note 37 for wash-
ing procedure). Ensure the wells are washed sufficiently by
completely filling them with wash buffer. It is important not to
let the wells dry out or enzyme activity will be lost.
2. Add 200 μL of blocking buffer and incubate overnight (or
alternatively for 1–2 h at 37 °C).
3. Thoroughly wash the plate again three times with the assay buf-
fer followed by three washes with PBS as before (see Note 37).

3.7.3 Primary Antibody 1. Add 100 μL of primary antibody (at the experimentally deter-
Binding Step mined optimal working concentration) to each well and incu-
bate for 5–6 h at room temperature (see Note 38).
2. Wash the plates as before (see Note 37).

3.7.4 Secondary 1. Add 100 μL of the diluted secondary antibody (at the experi-
Antibody Binding Step mentally determined optimal working concentration) to each
well and incubate at room temperature for 1 h.
2. Thoroughly wash the plates with assay buffer, as before (see
Note 37).

3.7.5 Detection Step 1. Detect bound antibody by the addition of a suitable substrate
solution to the wells (see Note 39).
2. As soon as the color develops add stop solution.
3. Read the plates immediately at A450 nm on a microtiter plate reader.
4. Draw the standard curve and estimate the protein concentra-
tion in the unknown sample.

3.8 Sodium Dodecyl SDS-PAGE is a widely used method for fractionating proteins
Sulfate- based on their mobility in an electric field. Binding of the deter-
Polyacrylamide Gel gent SDS confers different proteins with similar charge per unit
Electrophoresis mass ratios, and separation by PAGE is therefore based on protein
(SDS-PAGE) size. Many variations of this protocol have been described in the
of Proteins literature which become useful when the limits of the classical pro-
tocols are reached [22] (see Note 40). Instructions for use with an
ATTO protein gel electrophoresis system are laid out here and can
easily be adapted to other equivalent systems.
 Protein Quantitation and Analysis of Purity 241

1. Wash glass plates with detergent, rinse first with tap water, then
with dH2O and finally wipe in one direction with tissue soaked
in 100 % ethanol.
2. Place the gasket around the ridged plate, assemble the plates,
and secure with clamps.
3. Prepare a 10 % resolving gel by mixing 3.3 mL acrylagel,
1.35 mL bis-acrylagel, 2.5 mL 1.5 M Tris–HCl (pH 8.8),
2.61 mL dH2O, 0.10 mL 10 % (w/v) SDS, 0.10 mL 10 %
(w/v) APS (freshly prepared) and 0.01 mL TEMED.
4. Pour the gel to within 2 cm of the top of the larger plate (allow-
ing space for the stacking gel) and overlay with 100 % ethanol.
The resolving gel should be fully polymerized within 30 min.
5. Prepare the 5 % stacking gel by mixing 0.42 mL of acrylagel with
0.168 mL bis-Acrylagel, 0.312 mL 1 M Tris–HCl (pH 6.8),
1.55 mL dH2O, 0.025 mL 10 % (w/v) SDS, 0.025 mL 10 %
(w/v) APS and 0.0025 mL TEMED (see Note 41).
6. Remove the ethanol, pour the stacking gel, and immediately
insert a clean comb (wiped with 100 % ethanol). Allow the gel
to polymerize for at least 20 min.
7. Fill the electrophoresis tank with 1× Tris–glycine running buf-
fer to a level of about 5 cm deep.
8. After polymerization, remove the clamps and gaskets and lower
the prepoured gels into the buffer (place the gels into the buf-
fer at an angle to exclude air bubbles from the gel interface)
notched plate facing inward.
9. Fix the gel plates firmly in place using the pressure plates.
Completely fill the tank (including the chamber formed by the
inner plates) with 1× running buffer and carefully remove the
comb from the gel (see Note 42).
10. Load the sample into the wells. Include at least one well with a
prestained molecular weight marker.
11. Complete the assembly of the gel unit and connect to a power
supply.
12. Electrophorese at a constant current of 30 mA per gel until the
blue dye front has reached the bottom of the gel.
13. When complete remove the plates, separate, and process the
gel as required (see Subheading 3.9 or 3.10).

3.9 Staining While commercially available preparations for fluorescent staining

of SDS-PAGE Gels of SDS-PAGE gels are widely available, visible stains like Coomassie
Blue and Silver discussed here remain the most widely used for
routine visualization [23] since they do not require costly reagents
or expensive equipment for detection and quantification.
242 Eva M. Campion et al.

3.9.1 Coomassie Brilliant The dye Coomassie Brilliant Blue G-250 binds nonspecifically to vir-
Blue Staining of SDS-PAGE tually all proteins and is commonly used as a convenient stain for
Gels proteins following fractionation by PAGE. Gels are first soaked in a
solution of the dye, and any dye that is not bound to protein diffuses
out of the gel during the destain steps. This stain binds to proteins
more or less stoichiometrically, and so this method can be used when
relative amounts of protein needs to be established by densitometry.
1. After electrophoresis, disconnect the gel unit from the power sup-
ply and disassemble the apparatus. Carefully separate the plates
and remove and discard the stacking gel. Cut one corner from the
resolving gel to allow the orientation of the gel to be followed.
2. Immerse the gel in Coomassie blue solution for 30 min with
constant gentle agitation.
3. After staining, immerse the gel in destain with constant agita-
tion. Refresh the destain four or five times at 1 h intervals until
all of the background staining has been removed from the gel
(see Note 43).
4. An image of the gel in black and white can be captured using a
UV trans-illuminator (switched to white light only and using a
white tray), or alternatively the gel can be imaged or scanned
onto a computer (see Note 44).

3.9.2 Silver Staining Silver staining is a highly sensitive method for visualizing proteins
of SDS-PAGE Gels following electrophoresis. Detection of 0.3–10 ng of protein is
possible, making it up to 100 times more sensitive than Coomassie
Blue staining. Although sensitive, not all proteins stain equally
using this method, and the linear dynamic range is rather limited.
Silver staining is therefore not favored when quantitative protein
expression analysis is required. Commercially available silver stain-
ing kits that offer improved compatibility with subsequent mass
spectrometric work include SilverQuest™ Silver Staining Kit
(Thermofisher), Pierce™ Silver Stain for Mass Spectrometry,
ProteoSilver™ Plus Silver Stain Kit (Sigma-Aldrich), and PlusOne
Silver Staining Kit (GE Healthcare).
1. Following electrophoresis (see Note 45), disconnect the gel
unit from the power supply and disassemble the apparatus.
Carefully separate the plates and remove and discard the stack-
ing gel. Cut one corner from the resolving gel to allow the
orientation of the gel to be followed.
2. Place the gel in a clean container in fixing solution for 1–2 h
with gentle agitation at room temperature (for gels previously
stained with Coomassie blue, wash overnight with dH2O and
start from step 3).
3. Discard the fixing solution and wash the gel three times for
20 min each in wash solution.
 Protein Quantitation and Analysis of Purity 243

4. Incubate the gel in oxidizing solution for 10 min.

5. Remove the oxidizing solution and wash the gel for 10 min in
copious amounts of water. Repeat the washing procedure until
the pale yellow color has completely faded.
6. Soak the gel in silver nitrate solution for 30 min to stain.
7. Following staining, wash the gel twice for 2 min each using
plenty of water.
8. Place the gel in developing solution for 1 min. Replenish the
developing solution and incubate for a further 5 min. Repeat
this process until bands are stained satisfactorily.
9. Immerse the gel in stop solution for 5 min.
10. Store the gel at 4 °C in 1 % (v/v) acetic acid. See Note 44 for
analysis.

3.10 Western Western blotting (also known as immunoblotting) is a method

Blotting used to detect specific proteins, for which an antibody is available,
in a cell culture or tissue extract. Total cellular proteins are first
fractionated by gel electrophoresis under denaturing or non-
denaturing/native conditions. The proteins are then transferred to
a nitrocellulose or nylon membrane onto which they are then
immobilized (the blot). Primary antibodies (either monoclonal or
polyclonal) that are specific to the protein under investigation, are
then used to “probe” the blot. When bound to the blot, these
antibodies are then in turn located using a secondary antibody that
is labeled with a reporter enzyme such as a peroxidase (POD) or
alkaline phosphatase. Reporter enzyme activity is then detected by
incubation of the blot with an appropriate substrate, yielding a
detectable product that maps to the location of the protein of
interest. The use of fluorescently labeled secondary antibodies have
extended the dynamic range of quantitation [24] and have become
a popular alternative to reporter enzymes. Specific proteins can
thus be detected, even when present at very low levels in cell
extracts. The outcome of a Western blot experiment depends heav-
ily on the quality of the primary antibody available for the protein
under study, and there are now many companies that offer specific
antibodies against a vast array of protein targets, enzyme-conjugated
secondary antibodies, and detection kits. The user should adhere
to any recommendations made by the manufacturers of the appa-
ratus, antibodies, transfer membrane, and immunodetection kits
being employed. One should also survey any published literature
on the protein under study and take note of the reagents and
immunoblotting conditions that are most frequently described.
The directions given here are generic and assume the use of a Bio-
Rad Trans-Blot® SD semi-dry electrophoretic transfer cell.
244 Eva M. Campion et al.

3.10.1 Electrophoretic 1. Carry out SDS-PAGE as previously described (see Subheading 3.8).
Transfer of Proteins The use of commercially available prestained protein size markers
from PAGE Gels is recommended.
to Nitrocellulose Filters 2. Disconnect the gel unit from the power supply and disassem-
ble the apparatus. Carefully separate the plates and remove and
discard the stacking gel. Cut one corner from the resolving gel
to allow the orientation of the gel to be followed.
3. Soak the gel in transfer buffer for 15 min to equilibrate the gel
removing salts and detergents.
4. Cut the transfer membrane (e.g., nitrocellulose) to the same
dimensions as the gel, along with six pieces of 3MM filter paper
required to complete the gel/membrane sandwich.
5. Place a presoaked sheet of filter paper onto the platinum anode.
Roll a pipette over the surface of the filter paper to exclude all air
bubbles. Repeat this step with two more sheets of filter paper.
6. Place prewetted blotting membrane on top of the filter paper
and exclude all air bubbles as before (not all types of mem-
brane require prewetting).
7. Place the equilibrated gel carefully on top of the nitrocellulose
membrane, align the gel on the center of the membrane, and
ensure all the air bubbles removed.
8. Place another three sheets of prewetted filter paper on top of
the gel, again remove air bubbles (see Note 46).
9. Put the cathode on top of the stack and place the safety cover
on the transfer unit. Transfer the gel for 30 min at 17 V (trans-
fer conditions should be experimentally determined for opti-
mal results).
10. Once the transfer is complete, disassemble the apparatus. Leave
the gel in place on top of the nitrocellulose membrane so that
the membrane can be cut to the shape of the gel (including the
cut corner for orientation) using a sharp blade. The gel and any
excess membrane can then be discarded.
11. The colored molecular weight markers should be clearly visible
on the membrane.

3.10.2 Staining 1. Following electrophoretic transfer, immerse the nitrocellulose

of Proteins Immobilized membrane in 20 mL Ponceau S solution and stain for 5 min
on Nitrocellulose Filters with constant agitation.
2. After proteins are visualized, wash the membrane in several
changes of dH2O until all the excess stain has been washed
away. The loading of equal quantities of sample across all
lanes—or otherwise as the case may be—will be apparent. The
membrane can now be used for immunological probing.
 Protein Quantitation and Analysis of Purity 245

3.10.3 Immunological 1. Following Ponceau S staining, incubate the membrane in

Detection blocking buffer (see Subheading 2.10) for 3 h at room tem-
perature with constant agitation.
2. Discard the blocking buffer and quickly rinse the membrane prior
to the addition of the primary antibody (diluted appropriately in
blocking buffer) at 4 °C overnight on a rocking platform.
3. Remove the primary antibody (see Note 47) and wash the
membrane three times for 15 min each with 50 mL TBS-T [if
using conjugated primary antibodies then proceed to develop
the blot after these steps (i.e., go to step 6)].
4. Add the secondary antibody (diluted in blocking buffer) to the
membrane and incubate for 90 min at room temperature with
constant agitation.
5. Remove the secondary antibody and wash the membrane three
times for 15 min each with 50 mL TBS-T.
6. During the final wash, bring 2 mL aliquots of the appropriate
substrate (e.g., BCIP/NBT or TMB) to room temperature.
7. Place the membrane in a clean container and cover with the
substrate. (If using a chemiluminescent or fluorescent assay
then follow the manufacturer’s instructions accordingly).
8. When sufficient chromogenic development has occurred (usu-
ally within 10 min, but in some cases it may take several hours)
revealing bands of satisfactory intensity, rinse the membrane in
dH2O to stop the reaction.
9. Photograph or scan the blot.

3.11 qIPCR Assay Nucleic acid amplification methods can be used for signal genera-
tion in antibody-based immunoassays [25]. qIPCR is based on the
use of specific antibodies that have been conjugated to a nucleic
acid molecule that is targeted for amplification by real-time PCR,
the method of choice for quantitative determinations of low levels
of DNA. The benefits that are brought to bear include dramatically
improved limits of detection (by a factor of up to 10,000), the
capacity to work with small sample volumes, and assay formats that
are compatible with complex biological matrices [26].
Various synthetic DNA sequences and the corresponding PCR
primers may be used as a target region [27]. Several possible strate-
gies are available for linking antibody recognition with nucleic acid
amplification. These strategies have evolved to combine the advan-
tages of several of the latest technologies with those of qIPCR. The
pairing of a qIPCR assay with Phage display [28, 29] (where the
phage particle supplies both the detection antibody and the DNA),
use of asymmetric PCR [30] (for higher sensitivity of detection),
the incorporation of gold nanoparticles to allow easier coupling
between antibodies and DNA [31] and modified double-stranded
DNA molecules [32] have resulted in an “ultrasensitive” and
246 Eva M. Campion et al.

powerful technique with a detection limit—in at least one

instance—as low as 450 fmol/L [30]. Here, we describe the four
most widely used assay formats for the assembly of a qIPCR detec-
tion system. To further increase the sensitivity of the assay a com-
bination of the qIPCR assay with the technologies mentioned
above should be considered.
Assay Format (I): The capture antibody is first adsorbed to the
PCR tube surface and streptavidin is used to couple biotinylated
DNA to biotinylated detection antibody. Assay Format (II): The
capture antibody is first adsorbed to the PCR tube surface and
DNA-antibody conjugate is mixed with the antigen or sample
before addition to the well. Assay Format (III): Streptavidin-
coated microtiter plates are used, and capture antibody, DNA-
antibody conjugate and antigen, or sample are premixed before
addition to the well. Assay Format (IV): Antigen and samples are
adsorbed to the surface of the well prior to the addition of DNA-
antibody conjugate. All methods require some prior knowledge of
real-time PCR, and for the appropriate instrumentation and soft-
ware to be present in the laboratory [The procedure described
below employs the ABI 7500 and the corresponding 7500 soft-
ware (Applied Biosystems)].

3.11.1 Preparation 1. Use a 5′-biotinylated forward primer and an unmodified

of Biotinylated DNA Label reverse primer when using PCR to prepare biotinylated DNA.
2. Assemble the PCR component mixture in a total volume of
50 μL (see Note 48).
3. Perform PCR using the following temperature and time pro-
file: hold at 95 °C for 5 min (1 cycle); denature at 95 °C for
30 s, anneal at X °C for 30 s (X is the annealing temperature
specific to the primer set), extend at 72 °C for 40 s, (40 cycles);
then perform a final extension step at 72 °C for 5 min.
4. Analyze the PCR product obtained by standard agarose gel elec-
trophoresis in conjunction with appropriate DNA size markers.
5. Locate, excise, and purify the PCR product using a PCR prod-
uct Clean-Up kit according to the manufacturer’s instructions.
6. Concentrate the purified biotinylated DNA label using a G-50
Sephadex Column according to the manufacturer’s instructions.
7. Determine the concentration of the DNA label using UV-
visible spectrophotometry.
8. Aliquot appropriately and store at −20 °C. Biotinylated DNA
labels are stable at −20 °C for up to 1 year.

3.11.2 Preparation 1. Activate the antibody with thiol groups by mixing ~5 mg of anti-
of Antibody/DNA Label body (in PBS, 1 mM EDTA, pH 8.0) with 2-iminothiolane
Conjugate Traut’s reagent in tenfold molar excess in a total volume of 1 mL.
2. Incubate the reaction for 1 h at room temperature.
 Protein Quantitation and Analysis of Purity 247

3. Terminate the reaction with 30 μL of 1 M glycine-NaOH

pH 7.3.
4. Meanwhile, mix 40–50 nmol of amino-modified DNA (in
PBS, 1 mM EDTA, pH 7.2) with 2 mM of sulfo-SMCC in a
total volume of 500 μL.
5. Incubate for 30 min at room temperature.
6. Terminate the reaction with 13 μL of 1 M glycine–NaOH
pH 7.3.
7. Purify the activated antibody and the activated DNA label
using a 2 mL Zeba desalt spin column (Thermo-Scientific)
according to the manufacturer’s instructions.
8. Mix together and incubate on a shaker for 3 h at room
temperature.
9. Incubate overnight at 4 °C on a shaker.
10. Purify the conjugate using an ion exchange column (Resource Q
1 mL), according to the manufacturer’s instructions, using 10 mM
Tris–HCl pH 8.0 and elute with a 0–1.5 M NaCl gradient.
11. Pool fractions that contain the antibody/DNA conjugate and
concentrate to 0.3 mL using a Centricon YM-100.
12. Test the concentrated fraction by ELISA and qPCR (see
Subheadings 3.7 and 3.11.9).

3.11.3 Biotinylation 1. Pipette 2.5 mg/mL of detection antibody into a microfuge

of Detection Antibody tube.
2. Add BNHS (1 mg/mL in DMSO) in five to ten times molar
excess and mix gently but thoroughly.
3. Add one tenth volume of 1 M NaHCO3, pH 8.5 to the anti-
body solution.
4. Incubate at room temperature for 1–4 h.
5. Purify the biotinylated antibody either by dialysis (for 24 h
with three changes of PBS pH 7.4), or using a PD-10 gel filtra-
tion column according to the manufacturer’s instructions.
6. Store aliquots of biotinylated antibody at −20 °C (or under con-
ditions consistent with the stability properties of the antibody).

3.11.4 qIPCR Assay General comments: (1) In all assay formats set up a no antigen
Set Up control (to control for unspecific binding of antibody and DNA to
the surface), a negative sample control (to determine that the sam-
ple matrix does not give rise to a signal) and a no antibody control
(to check for any unspecific binding between antibodies). (2)
Prepare a standard curve by making a dilution series of antigen
(0.1–10 μg/mL) including 6–7 concentrations. Vortex and spin
between every dilution. (3) Set up two or three replicates of all
standards and unknowns that are to be assayed.
248 Eva M. Campion et al.

3.11.5 Assay Format I 1. Add 25 μL of diluted capture antibody to a microtiter plate

and incubate overnight on microtiter plate shaker at 4 °C.
2. Wash the wells three times with wash buffer (see Note 37).
Ensure the wells are washed sufficiently by filling the wells with
wash buffer.
3. Add 200 μL of blocking buffer and incubate for 1 h at 37 °C.
4. Wash the wells three times with wash buffer.
5. Set up dilutions of the antigen standard and the unknown sam-
ple across a concentration range of 0.1–10 μg/mL in the anti-
gen-coating buffer.
6. Add 25 μL of antigen or unknown sample to each well as
appropriate and incubate at room temperature for 1 h.
7. Wash the wells three times with wash buffer.
8. Add 25 μL diluted biotinylated detection antibody and incu-
bate for 1 h at room temperature.
9. Wash the wells six times with wash buffer.
10. Incubate with 25 μL streptavidin (5 nM) for 30 min at room
temperature.
11. Add 25 μL of the diluted biotinylated DNA label and incubate
at room temperature for 1 h.
12. Wash the wells six times with wash buffer.
13. Wash six times with ultrapure water.
14. Quantify using qPCR (see Subheading 3.11.9).

3.11.6 Assay Format II 1. Add 25 μL of the diluted capture antibody to a microtiter plate
and incubate overnight on a microtiter plate shaker at 4 °C.
2. Wash the wells three times with wash buffer (see Note 37).
Ensure the wells are washed sufficiently by filling the wells with
wash buffer.
3. Add 200 μL of blocking buffer and incubate for 1 h at 37 °C.
4. Wash the wells three times with wash buffer.
5. At the same time, dilute the antigen standard and the unknown
sample in a concentration range of 0.1–10 μg/mL in the
antigen-coating buffer.
6. Mix the antigen or sample together with the diluted chemically
conjugated antibody and DNA label. Incubate for 1 h at 37 °C.
7. Add 25 μL of this mix to the wells and incubate for 30 min at
room temperature.
8. Wash the wells six times with wash buffer.
9. Wash six times with ultrapure water.
10. Quantify using qPCR (see Subheading 3.11.9).
 Protein Quantitation and Analysis of Purity 249

3.11.7 Assay Format III 1. Prepare dilutions of the antigen standard and the unknown
sample in the antigen-coating buffer across a concentration
range of 0.1–10 μg/mL.
2. Block the streptavidin-coated microtiter plates with 200 μL
blocking buffer for 1 h at 37 °C.
3. At the same time, mix 5.5 μL antigen or sample with 11 μL of
diluted chemically conjugated antibody and DNA label, and
11 μL of the diluted biotinylated capture antibody. Incubate
this mix at room temperature for 1 h.
4. Wash the wells three times with wash buffer (see Note 37).
5. Add 25 μL of the mix to the wells and incubate for 30 min at
room temperature.
6. Wash the wells six times with wash buffer.
7. Wash six times with ultrapure water.
8. Quantify using qPCR (see Subheading 3.11.9).

3.11.8 Assay Format IV 1. Dilute the antigen standard and the unknown sample in a con-
centration range of 0.1–10 μg/mL in antigen-coating buffer.
2. Add 50 μL to each well.
3. Incubate the plates on microtiter plate shaker, overnight at 4 °C.
4. Wash the wells three times with wash buffer (see Note 37).
5. Add 200 μL of blocking buffer and incubate for 1 h at 37 °C.
6. Wash the wells three times with wash buffer.
7. At the same time, mix the antigen or sample together with the
chemically conjugated antibody and DNA label. Incubate for
1 h at 37 °C.
8. Add 25 μL of the mix to the wells and incubate for 30 min at
room temperature.
9. Wash the wells six times with wash buffer.
10. Wash six times with ultrapure water.
11. Quantify using qPCR (see Subheading 3.11.9).

3.11.9 Real-Time PCR 1. Prepare the reagents for the PCR reaction as a master mix
(qPCR) Assay (make up 10 % more than the volume required so as to account
for potential pipetting error).
2. Include three “no template controls” (NTC, a minus sample
control) in each reaction plate. Set up a positive control sample
using DNA/Conjugate as template.
3. Aliquot 20 μL of PCR master mix per reaction into the indi-
vidual wells of the PCR plate containing the immobilized DNA.
4. Immediately seal the plate with an optical adhesive cover and
protect from light. Centrifuge at 500 × g for 2 min to eliminate
air bubbles. It is important to check to ensure that no bubbles
remain in any well.
250 Eva M. Campion et al.

5. Perform amplification and detection under the following

cycling conditions: hold at 50 °C for 2 min (1 cycle); hold at
95 °C for 5 min (1 cycle); denature at 95 °C for 20 s, anneal at
X °C for 20 s (X is the annealing temperature specific to the
primer set), extend at 72 °C for 25 s (45 cycles).
6. Record fluorescence data during the 72 °C step.
7. Include a dissociation stage to analyze PCR product melting
temperature.
8. Analyze the qPCR data obtained with the appropriate soft-
ware. Check for bimodal dissociation curves or an abnormal
amplification plot.

4 Notes

1. When selecting the method to be used for total protein deter-

mination, care should be taken to consider sample and buffer
properties and the objective of the analysis being undertaken.
The advantages and limitations of the most commonly employed
methods are compared in Table 1, as an aid to the user in the
selection of the most suitable assay for the sample in question.
2. All solutions should be stored at room temperature prior to
the assay (cold solutions can cause condensation on the surface
of the cuvette, whereas warm solutions often lead to bubbles
causing inaccuracy in the readings).
3. The selection of a protein standard is an important step in
every protein assay as the standard and sample should be of a
similar molecular weight and should respond in the same way
to the assay in order to minimize errors in estimations.
4. Protein concentrations are measured in milligrams per millili-
ter (mg/mL) and micrograms/microliter (μg/μL).
Concentrations can also be recorded in micrograms/milliliter
(μg/mL) for very small concentrations.
5. Common buffer components such as acetate, Brij 35 deter-
gent, deoxycholate, SDS, Triton X-100, Tween 20 and urea
are known to absorb strongly at 280 nm and should be avoided.
Nucleic acid contamination of samples can also greatly influ-
ence the absorption.
6. Common buffer components such as acetic acid, ammonium
sulfate, 2-glycerol, mercaptoethanol, Tris, and SDS are known
not to be compatible with the Bradford Assay.
7. NaOH is added to ensure that the sample does not precipitate
upon addition of Bradford reagent.
8. During storage dye may precipitate from solution. If this
should happen filter the reagent before use.
 Protein Quantitation and Analysis of Purity 251

9. Quartz cuvettes cannot be used as the Coomassie brilliant blue

G-250 dye binds to this material.
10. Many substances are known to interfere with the Lowry pro-
tein assay including CAPS, barbital, cesium chloride, EDTA,
citrate, cysteine, diethanolamine, dithiothreitol, EGTA,
HEPES, mercaptoethanol, nonidet P-40, phenol, sodium
deoxycholate, polyvinyl pyrrolidone, sodium salicylate, thimer-
osol, tricine, Tris, and Triton X-100.
11. Substances known to interfere with the BCA assay include
Tris, ammonium sulfate, EDTA, DTT, EGTA, and SDS.
12. Commonly used blocking agents include BSA, serum, nonfat
dry milk, casein, and gelatin. The selection of blocking agent is
dependent on the antibody being used (see the manufacturer’s
guidelines).
13. Allow wash buffer to equilibrate to room temperature before use.
14. Nonfat dried milk is not compatible for use with streptavidin/
biotin.
15. For accuracy, prepare standards that span the full range of the assay.
16. Use ultrapure water for preparation of all buffers.
17. Optimal concentrations are determined experimentally or fol-
lowing manufacturer’s guidelines.
18. A plate material that can bind biological materials should be
used. For samples containing low concentrations of target anti-
gens, the adsorption to reaction vessels can be minimized using
silanized cups. PCR plates can also be immersed in 0.8 % glu-
taraldehyde solution at 37 °C for more than 6 h in order to
improve absorbability.
19. UNG is included in the PCR to prevent the reamplification of
carryover PCR product. Through the use of dUTP rather than
dTTP, the uracil incorporated into amplicons during PCR can
be destroyed by UNG, thus destroying any contaminating
PCR product present at the outset. The UNG incubation step
involves incubating reactions at 50 °C for 2 min and precedes
the HotStart Taq DNA polymerase activation step (95 °C for
5 min). UNG is then thermally inactivated during the first
denaturation step of the PCR.
20. Concentrated protein solutions should be diluted so they are
within the linear range of the instrument being used. If the
approximate sample concentration is unknown, a range of
dilutions (1, 1:10, 1:100, 1:1000) should be assayed. When
working with dilute samples, the addition of a non-ionic deter-
gent to the buffer may help to prevent loss of protein through
adsorption of protein on the cuvette.
21. If a clear linear relationship is observed, a standard curve is not
necessary. Amounts can be determined using interpolation.
252 Eva M. Campion et al.

However, a standard curve should be prepared to check for

accuracy and linearity the first time that an assay is performed.
22. Choose the appropriate protein reference (e.g., BSA, IgG, and
lysozyme) for calculations of protein concentration in order to
obtain the desired molar extinction coefficient.
23. It is recommended to use a precision pipette (0–2 μL) with
precision tips to ensure that sufficient sample (1–2 μL) is deliv-
ered to the measurement pedestal.
24. For Bradford, Lowry or BCA assays, or when working with
purified protein a 2 μL sample size will ensure proper column
formation.
25. It is recommended that an aliquot of the blanking buffer be mea-
sured as a sample to confirm that the instrument is working well
and that dried-down sample on the pedestals is not a concern.
26. A microassay format can be used for protein concentrations
between 1 and 20 μg/mL. The protocol followed is the same
as that of the basic protocol except with a reduced total vol-
ume of 1 mL. The assay responds primarily to arginine resi-
dues, and an arginine-rich standard may be preferable if the
sample is rich in that amino acid.
27. The sample can be diluted in the case of a high protein
concentration.
28. The standard curve is not linear, and the precise absorbance
varies depending on the age of the assay reagent. As a result, it
is important to construct a calibration curve for each assay
performed.
29. The extent of color yielded in this assay is dependent on the
amino acid composition of the sample. Thus, two different
proteins at the same concentration can give significantly differ-
ent color yields. Hence the choice of a standard similar in com-
position to that of the sample is an important consideration in
the design of this assay. If this is not possible for any given assay
a normalized protein quantitation model based on the target
protein sequence has also been suggested [33].
30. This incubation period is not a critical parameter and can vary
from 10 min to several hours with no effect on final absor-
bance values.
31. A modification of the Lowry assay, using a microwave oven,
has been described that allows protein determination with
much reduced incubation times of just seconds [34].
32. If the A660 values are low then reread the samples at A750, which
may increase the sensitivity of the assay, or at A550 if the sample
concentration is between 100 and 2000 μg/mL.
33. Since this assay is not linear at high concentrations, ensure that
the sample falls on the linear portion of the standard curve.
 Protein Quantitation and Analysis of Purity 253

34. A modification of the BCA assay, utilizing a microwave oven,

has been described that allows protein determination with
much reduced incubation times of just seconds [34].
35. This can easily be achieved by placing the samples in a water
bath at room temperature.
36. Increase the incubation time in order to improve the sensitivity
of this assay. The incubation temperature should be lowered to
ambient room temperature to obtain a decrease in sensitivity.
37. Washing procedure:
Wash Step: use a squirt bottle/multichannel pipette to fill
the wells with wash solution. Do not touch the inside surface
of the wells with the pipette tips/or bottle nozzle. Expulsion of
wash solution: holding the microtiter plate firmly (from the
underneath) over a sink, turn the plate rapidly and directly
upside down causing the liquid to be forced out of the wells
and into the sink. Repeat this “dumping motion” a second
time. Blotting and banging the plate: immediately blot the
upside down plate (wells face down) on blotting/tissue paper.
Move the plate to an unused section of the blotting paper and
allow to drain for 5–10 s. Repeatedly “bang” the plate very
hard four to five times over unused areas of the paper. Wash
step: Use a squirt bottle/multichannel pipette to refill the wells
with wash solution as before and immediately dump and bang
the plate again as described above. Following the last wash,
leave the plate upside down for 30 s to drain. Bang again for
three to four times, rotating the plate between each bang.
38. This incubation time can be optimized.
39. Do not add substrate near the sink as the washing procedure can
create aerosols which could recontaminate the wells or substrate.
40. Chemical variations possible with this protocol include changes
to the recipes for gel structure, buffers, and detergents.
41. It is possible to include a small amount of bromophenol blue
in the stacking gel mix to give the gel a pale blue color. This
allows for easier visualization and loading of the wells [35].
42. Use a 3 mL syringe fitted with a 22-gauge needle to wash the
wells with running buffer and remove any unpolymerized gel.
Straighten wells using a loading tip.
43. Extensive destaining can lead to the loss of some low molecular
weight bands and the fading of other bands. Note: when stain-
ing IEF gels with Coomassie Blue, the gel must first be fixed in
a solution of trichloroacetic acid. This leaches out the carrier
ampholytes, which would otherwise produce background
staining. When staining small peptides (less than 10 kDa), the
gel is first fixed in a solution containing glutaraldehyde to
cross-link the peptides and prevent them from diffusing out of
the gel during subsequent staining steps.
254 Eva M. Campion et al.

44. Analysis of SDS-PAGE Gels: Determine the correct orientation of

the gel, using the cut corner to identify the top/bottom and left/
right ends. Locate the lanes corresponding to each sample loaded
on the gel. Identify the polypeptide bands of interest. Estimate
the approximate molecular mass or relative molecular mass for
each band of interest using the molecular weight markers as a
guide. Note differences in the intensity of band staining. This may
be indicative of disparity in abundance between individual poly-
peptides. Identify unusual patterns that might reflect incom-
plete denaturation or degradation of the proteins being
analyzed (overloading or underloading, distortion of lanes,
smears, and streaks can all limit the interpretation of results).
45. Maintain a high level of cleanliness during electrophoresis and
silver staining to minimize spurious artifacts. Wear gloves to
prevent staining of the gel due to finger marks.
46. Ensure that the nitrocellulose membrane is between the gel
and the anode or the proteins will be lost from the gel into the
buffer rather than transferred to the nitrocellulose.
47. Diluted antibody can usually be retained and stored at −20 °C
for a further four to five uses.
48. Use filter/barrier tips to avoid cross-contamination during
pipetting. Do not touch the microplate wells with the tips dur-
ing pipetting.

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