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Protein Calculation

This document provides instructions for calculating the concentration of protein in a sample using a standard curve method. You first need to select an appropriate protein quantification method and construct a standard curve by measuring absorbance values of protein standards of known concentration. From the standard curve, you obtain the equation relating absorbance to concentration. You then measure the absorbance of your sample and plug it into the equation to calculate the sample's protein concentration. An example calculation is provided.

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Irfan Salim
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137 views

Protein Calculation

This document provides instructions for calculating the concentration of protein in a sample using a standard curve method. You first need to select an appropriate protein quantification method and construct a standard curve by measuring absorbance values of protein standards of known concentration. From the standard curve, you obtain the equation relating absorbance to concentration. You then measure the absorbance of your sample and plug it into the equation to calculate the sample's protein concentration. An example calculation is provided.

Uploaded by

Irfan Salim
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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 You can use this formula to calculate the amount of protein in your sample:

[ (OD of the test sample) / (OD of the standard sample) ] X concentration of the
standard sample.
for example: If the OD of your test sample is 0.04 and the OD of your standard sample
(5 mg/ml) is 0.05. Then the amount of protein in your sample can be calculated as:
(0.04 / 0.05) X 5
=0.8 X 5
= 4.0 mg/mL protein (in test sample).
Regards,
A K Dubey

In simple terms you need to see the equation Y=mX+C, which can be generated by
plotting the std conc in Xaxis and absorbance in Y. You will get a linear line and excel
can generate the equation with m value and c value.
Once you have the equation, you can calculate X (bcos Y is your abosrbance value),
using the above equation. X= (Y-c)/m. here X is the unknown protein concentration.
I will tell you how to do with an example.Consider these are the values of the std curve
std conc absorbance
0 0.1545
2 ug 0.3565
4ug 0,463
6ug 0.602
8ug 0.658
10ug 0.708
unknown 0.512
So you need to plot a graph with std conc on X-axis and absorbance value in the Y-axis.
1.In excel , the charts you will find different type of graphs choose Scatter in that
marked scatter. Now you can see your graph.
2.Right click one of the points plotted in the graph and click add trend line
3. A new dialogbox will open, in that select Display equation on chart.
4. You will get an equation. This equation will give your unknown sample concentration.
The equation will be in this format. Y= mX+C (eg Y=0.0545X+0.218).
Here X is the unknown concentration which you need to find. Y value is the absorbance
that you got for your unknown sample.
Therefore X= (Y-C)/m. i.e X= (Y-0.218)/0.0545.
All the best. It looks long for the first time, but once you do it, then it is the easiest

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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