Many methods are used to calculate protein concentration such as, bicinchoninic acid assay, Bradford protein assay and the absorbance method at 280nm. These methods are based on the color change in the solution containing protein. This article on how to calculate protein concentration will explain all these methods in detail and help you in determining the protein concentration in a given solution.
Bicinchoninic acid assay
- This assay is also known as BCA assay which is completed in two steps. It determines the total protein concentration in a solution. This is determined by the change in color of the sample solution from green to purple. It is measured using colorimetric techniques.
- In this assay, in the first step Cu2+ ions from the cupric sulfate (one of the ingredients in a stock BCA solution) is reduced to Cu1+, forming a complex with biuret reaction.
- In the second step BCA forms a purple complex with Cu1+ at 526nm.
Requirements:
Reagent A:
- 1g bicinchoninate (BCA)
- 2g sodium carbonate
- 0.16g sodium tartrate
- 0.4g NaOH and 0.95g sodium bicarbonate.
To make the solution up to 100ml, mix reagents in 80ml distilled water and adjust pH to 11.25 with 8M NaOH
Reagent B: 4 % CuSO4.5H2O
- Now mix 50 volumes of reagent A with 1 volume of reagent B to make working solution or WS which is green in color and can be used till one week.
Procedure:
- Step 1: Take any two samples of the protein and prepare a diluted solution of them. Prepare the solution in such a manner that each 100µl of the solution contains 100µg of protein.
- Step 2: In each of the 100µl solution add 2 ml of working solution.
- Step 3: Seal the sample and incubate them at 60 degrees of temperature for 15 min.
- Step 4: Now, cool the samples and measure the absorbance at 562nm, subtract the average values at 562nm blank from the 562nm measurements.
- Step 5: Plot a graph of standard vs. concentration and study the slope of this curve to estimate the protein concentration.
Bradford assay
Requirements:
- BSA or bovine serum albumin
- 0.15 M NaCl
- Coomassie brilliant blue 1
- Spectrophotometer, micropipettes and tubes
Procedure:
- Step 1: Prepare protein standards using diluted BSA with 0.15 M NaCl to the concentrations of 0, 250, 500, 750 and 1500 µg BSA/mL.
- Step 2: Prepare dilutions of the samples whose protein concentration is to be measured.
- Step 3: Add 100 µL of each of the above solutions in the test tubes.
- Step 4: Add 5.0 mL of Coomassie Blue to the test tubes and mix them using the vortex.
- Step 5: Adjust the spectrophotometer to 595 nm wavelength using the blank solution or 0 BSA.
- Step 6: Wait for some time and read each of the standards and each of the sample readings.
- Step 7: Plot the graph of the absorbance of standards against their concentration, determine the extinction coefficient and calculate the concentrations.
Absorbance at 280nm
This method is used when you know the sequence of protein in the given sample. This is a theoretical method of calculating the protein concentration. Here we calculate the extinction coefficient at a wavelength of 280nm using the equation given below,
Ε280nm (M-1cm-1) = (#Try)(5500)+ (#Tyr)(1490)+(#cystine)(125)
Procedure:
- Step 1: Calculate the coefficient of extinction using the above equation.
- Step 2: Now, warm up the UV lamp for about 15 min.
- Step 3: Set the spectrophotometer to zero to buffer at wavelength of 280 nm in quartz cuvette.
- Step 4: Measure the absorbance of protein solution.
- Step 5: Calculate the protein concentration using the equation given below,
[Protein] (Mg/mL) = A280nm / (Ε 280nm x (cuvette path length in cm))
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