Question

How to calculate amount of protein in bradford assay?

Answer 1

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First you have to select the best method for measuring protein, according to sample type and presence of substances as potential interferences.

The Bradford method, Lowry method, Absorbance at 280 nm method, have their advantages and disadvantages.

Bradford method is fast, sensitive, accurate, has low interferences.

The absorbance at 280 method is fast, requires no chemicals, is non-destructive, but is low sensitive and inaccurate.

The method of Lowry has many steps, the color is dependent on the incubation time, it is less sensitive than Bradford and has a lot of interference with common substances.

In any case you must construct a standard curve (using bovine albumin).

For that you have to prepare a stock solution of albumin, and from this prepare different solutions of known concentration.

These albumin solutions are used as samples, applying the selected method, to construct the standard curve.

Graph Absorbance vs concentration, and obtains the equation of the line (y = mx + b), with r2, as close to 1 as possible.

Another option is to subtract the value of "zero" protein, then the straight line passes through zero and the equation simplifies: y = mx.

Where y is the absorbance, m is the slope and x is the concentration.

Thus, x = y / m.

The absorbance of the sample is divided by the value of the slope and the concentration is obtained.

If the sample was diluted, the value is adjusted by multiplying by the dilution factor.

Example:

Absorbance = 0. 36

Sample was diluted 10 times

The absorbance of the "zero" is 0.053

Adjusted absorbance: 0. 360-0.053 = 0.307

The standard curve equation: y = 0.012x

x = y / m,  therefore x = 0.307 / 0.012

x = 25.58 ug / ml

But sample diluted 10 times:

x = 25.58 x 10 = 255.8 ug prot / ml

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