Protein Quantification Techniques Report
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ScribdTranslationsProtein Quantification Techniques Report
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ScribdTranslationsREPORT OF PRACTICE # 2
Quantification of proteins
Members:
Acosta Fernández Ilya Fabiola, Luna Vargas Tiaret, Reyes Emmanuel Cristian
Alejandro, Torres López Nallely Paola.
08/11/2021
Molecular Biology of the Cell I
Group: 5045
Felipe Alcantara Sanchez
Introduction
The quantification of proteins in an experiment can be used to measure the
biomass, measure the performance throughout a purification process, to measure the
specific enzymatic activity (which is expressed as speed per mass of
protein used); it is also a requirement for other analytical procedures such as the
purity evaluation, amino acid sequencing, spectrometry of
masses, etc. In a biochemistry laboratory, for the quantification of proteins, one
they use various spectrophotometric techniques based on the interaction of the
light with matter.
UV-visible spectrophotometry is a technique that allows determining the
concentration of a compound in a solution. Its basis is that the
molecules absorb electromagnetic radiation and the amount of light absorbed
It will depend on the concentration. For this method, a spectrophotometer is used.
in which the wavelength of the light that passes through can be selected
solution and measure the amount of light absorbed by it.
Colorimetric methods, which are based on a
reagent or dye that binds to proteins, and with this there is a change in the
coloration of the examined substance.
The Biuret method is based on the formation of a colored complex between the
Cu2+ and the NH groups of the peptide bonds in basic medium. 1Cu2+ is
it complexed with 4 NH. (Fernandez 2010, p. 3) The intensity of the coloration is
proportional to the amount of proteins. Although the sensitivity of this method is
very low so it is only recommended for the quantification of proteins in
very concentrated solutions, like serums. The violet color is read at 540nm
against standard BSA curve and the intensity of the color is proportional to the amount of
protein
The Bradford method is based on the binding of a dye, Coomassie Blue G-250.
(also Serva Blue) to the proteins (Fernandez 2010, p. 3). It is a method
sensible, simple, fast and cheap and there are few substances that interfere in the
determination of proteins. It is based on the binding of the Comassieblue reagent
G250 to proteins in an acidic medium, recognizes amino acids such as arginine,
phenylalanine, tryptophan, and proline also produce a deep blue color at A=595nm
In the BCA method, bicinchoninic acid is used, which is a compound capable
to form Cu1+ ions in alkaline medium. In this method, the ion is monitored
cuprous that is produced in a reaction between proteins and Cu2+ in a medium
alkaline. As a result, there is a method that is simple, rapid, and very sensitive,
in addition to tolerating various compounds that could alter the previous ones
methods. It is based on the fact that proteins reduce cupric ions from the BCA reagent.
to cuprous ions in a basic medium. A color change from green (BCA) is observed.
the purple.
Finally, the Lowry method combines the Biuret reaction with the reduction of
FOLIN-CIOCALTEAU reagent for the aromatic groups of tyrosine residues,
tryptophan, cystine cysteine and histidine. From this, a blue compound is formed.
intense and measures 750nm.
For this practice, the purification of the milk protein was previously carried out.
by means of precipitation with acid from a sample of skim milk, one
They obtained 6.33 g of a cream-colored powder from 200 mL of milk.
The objective of this practice is to quantify the protein concentration in milk.
the serum by means of a Biuret test, for this serum albumin will be used
bovine (BSA) as reference protein.
Objective: To know the spectrophotometric techniques and to determine the concentration of the
protein that we were able to extract from milk.
Hypothesis: How much protein concentration can we obtain after doing
the necessary calculations?
Materials and methods
To carry out the procedure, a series of steps were followed for the
quantification, starting with washing 20 test tubes to subsequently
label them in 3 series from 1-6 and arrange them in a rack, the two tubes
Leftovers were labeled as 'problem sample 1' and 'problem sample 2'.
Subsequently, a stock of BSA was prepared at a concentration of 20 mg/mL and was
they prepare serial dilutions of the reference protein (BSA) in triplicate at a
final volume of 1.0 mL. The concentrations of each tube must cover the range
of sensitivity of the assay that will be followed. For the biuret assay, from 1 to 15
mg/mL is appropriate. For this exercise, the following concentrations were used.
final concentrations: 2, 5, 8, 10 and 12 mg/mL. The volume of the stock (V1) for each
dilution and the volume of water necessary to complete the milliliter is calculated by
subtraction. This data was documented in the following table.
Concentration Necessary volume of
BSA final number Necessary volume water to complete 1.0
tube (mg/mL) from stock (mL) mL
1 0 0 1
2 2 0.1 0.9
3 5 0.25 0.75
4 8 0.4 0.6
5 10 0.5 0.5
6 12 0.6 0.4
To continue with the experiment after documenting the data, a pipette is used.
sample of each dilution, 1.0 mL of undiluted serum and 1.0 mL of 10% diluted milk
A recommended volume of reagent is added to one tube at a time.
laboratory manuals with an exclusive pipette for this reagent and it is stirred
immediately until achieving a uniform mixture and avoiding the formation of foam. To
mix, it is recommended to use a vortex at medium-high speed for 10 seconds and
after returning the tube to its place in the rack, it is allowed to rest at temperature and
recommended time. Then the spectrophotometer is turned on so that
heat the lamp and adjust the wavelength (λ) in the spectrophotometer
required. For biuret, it is read at 545 nm, which is the wavelength of the maximum
of absorption of the complex called biuret. The appropriate volume of the tube is poured.
white to a cuvette or bucket for spectrophotometer and placed correctly
inside the device, so that the light passes through the sample without
interference. The target is configured on the equipment so that it appears on the display.
Abs 0.000y is poured into a second bucket, the first shows for reading to
note the numerical value shown If the value changes constantly, the
the sample is not homogeneous or the reaction continues. Another sample is prepared. If the
the value shown on the display is negative, that means that the concentration of the
analyte is below the sensitivity limit of the assay and is recorded as 0
mg/mL or, as "less than the limit of sensitivity". If the value is close to or greater than
at 2, the sample is too concentrated and exceeds the sensitivity limit of
essay. Immediately after each reading, the sample must be removed from the
bucket and rinse several times with distilled water or 70% ethanol.
Finally, the equipment is turned off and disconnected, it is cleaned with a damp cloth and
waste is discarded in the designated containers.
Results
Table 1. Absorbance values at 545 nm (A545of the BSA dilutions
A545 Protein
BSA (mg/mL) e1 e2 e3 average (mg/mL)
0 0 0 0 0 0
2 0.076 0.044 0.078 0.066 2
5 0.135 0.125 0.129 0.130 5
8 0.195 0.162 0.195 0.184 8
10 0.263 0.241 0.221 0.242 10
12 0.314 0.305 0.26 0.293 12
Table 2. Absorbance at 545 nm of the problem samples with biuret reagent
Vol. [*] from
Sample Vol. H2O Total Vol. protein
Sample (mL) (mL) (mL) A545 (mg/mL)
Milk 0.1 0.9 1 0.087 3.37 mg/0.1mL
Serum 1 0 1 0.128 5.12 mg/1mL
Pattern curve
Discussion and conclusions
As can be seen in table 1, the average absorbance is
proportional to the concentration of protein as had been speculated.
And from the values obtained in Table 1, an average can be obtained.
the concentrations of BSA which will allow us to establish a standard curve, of the
how we can obtain an equation that will indicate the correlation that exists
between the line and the results we obtained, due to the correlation
the line R has a value close to 1, our relationship is good, so
we could isolate X from our equation and substitute it with any value of
absorbance in order to obtain the concentration of proteins in a more
needs.
As such, we could not obtain results, since the session was purely online.
therefore the results we used were those shown in class.
Bibliography
Fernández Reyes, Emilio and Aurora Galván Cejudo. 'Methods for Quantification'
of proteins". Department of Biochemistry and Molecular Biology, 2010.
Determination of proteins
The provided text is a URL and cannot be translated..
Classes with Felipus. "Quantification of proteins". YouTube, November 5
2021. Video, 33:30. [Link]
