Biochemistry Lab
Experiment 5: Total Protein Estimation by Lowry’s Method
Report 5
Objective
To determine the concentration of proteins by Lowry’s method.
Reagents Required
1. BSA stock solution(1mg/ml),
2. Analyticalreagents:
(a) 50 ml of 2% sodium carbonate mixed with 50 ml of 0.1 N NaOHsolution (0.4 gm in 100 ml
distilledwater.)
(b) 10mlof1.56%coppersulphatesolutionmixedwith10mlof2.37%sodiumpotassiumtartarate
solution. Prepare analytical reagents by mixing 2 ml of (b) with 100 ml of(a)
3. Folin - Ciocalteau reagent solution (1N) Dilute commercial reagent (2N) with an equal volume of
water on the day of use (2 ml of commercial reagent +2 ml distilled water)
Principle
The phenolic group of tyrosine and tryptophan residues (amino acid) in a protein will produce a
blue-purple color complex, with maximum absorption in the region of 660 nm wavelength, with
Folin- Ciocalteau reagent which consists of sodium tungstate molybdate and phosphate. Thus the
intensity of color depends on the amount of these aromatic amino acids present and will thus
vary for different proteins. Most proteins estimation techniques use Bovin Serum Albumin
(BSA) universally as a standard protein, because of its low cost, high purity and ready
availability. The method is sensitive down to about 10µg/ml and is probably the most widely
used protein assay despite its being only a relative method, subject to interference from
Trisbuffer, EDTA, nonionic and cationic detergents, carbohydrate, lipids and some salts. The
incubation time is very critical for are producible assay. There action is also dependent on pH
and a working range of pH9 to 10.5 is essential.
Procedure
1. Different dilutions of BSA solutions are prepared by mixing stock BSA solution(1mg/ml) and water
in the test tube as given in the table. The final volume in each of the test tubes is [Link] BSA
range is 0.05 to 1 mg/ml.
2. From these different dilutions, pipette out 0.2 ml protein solution to different test tubes and add 2ml
of alkaline copper sulphate reagent (analytical reagent). Mix the solutionswell.
3. This solution is incubated at room temperature for 10mins.
4. Then add 0.2 ml of reagent Folin Ciocalteau solution (reagentsolutions) to each tube and incubate
for 30min. Zero the colorimeter with blank and take the opticaldensity (measuretheabsorbance) at
660 nm.
5. Plot the absorbance against protein concentration to get a standard calibrationcurve.
6. Check the absorbance of unknown sample and determine the concentration of the unknown sample
using the standard curve plottedabove.
Sample conc. Sample vol Alk. CuSO4 Lowry reagent O.D.
(mg/ml) (ml) (ml) (ml) 600 nm
0.114
0.05 0.2 2 0.2
0.1280
0.1 0.2 2 0.2
0.4341
0.2 0.2 2 0.2
0.4825
0.4 0.2 2 0.2
0.7374
0.6 0.2 2 0.2
0.8927
0.8 0.2 2 0.2
1.0186
1.0 0.2 2 0.2
***Draw standard curve to determine the unknown concentration of proteins (linear equation and R 2 should
be represented in standard curve)
Y-Values
1.2
f(x) = 1.11797514124294 x
1 R² = 0.977548026789314
0.8
Y-Values
Linear (Y-Values)
0.6
0.4
0.2
0
0 0.2 0.4 0.6 0.8 1 1.2
******Write short notes on the following points:
1. Beer-Lambertslaw
For unknown concentration (with absorbance of 0.4341)
A=abc
a= slope= 1.118
b=1 cm
c=unknown
A= 0.4341 (given)
So c=A/a= 0.4341/1.118= 0.388 mg/ml
2. Compare Folin-Lowry method with other methods of protein estimation.
Its advantage is that it is a sensitive assay which requires no digestion of protein, it is 10 or 20 times more sensitive as
compared with ultraviolet absorption at 280 nm, it is more specific and less interrupted by turbidity, it is significantly
more sensitive than the ninhydrin reaction and burette reaction, it is simple to perform and can be easily used on small
scale in the labs.
Its disadvantages are that the amount of colour developed differs from protein to protein, it is less constant than the
biuret reaction, but more constant than the absorption at 280 nm. The color is not exactingly proportional to
concentration.
