Hi, 

That very simple, you need to use microsoft excel.

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

Dear Heba Huessin,

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

How to calculate the M and C values

Very good answer from Mohammad .

This is if the standard curve is linear, however, with BSA standard curve (x axis is time starting from 0) it is not linear and is not that simple. I'm wondering if anyone have used the solver in excel for this?

Thanks

Very good answer. Really appreciated

Very nice explanation by Mohammad

I did my calculations the same way however, for one of the unknown solutions, I got a concentration of 1.23. I know the Lowry test determines protein concentrations of 0.01-1.0up/ml so I was wondering if this is possible or something went wrong during the experiment or calculations.

The relationship between concentration and absorbance is not linear in BCA/ELISA assays.

You can use Excel Trendlines to fit Polynomial relationships.

All clearly explained in

https://tools.thermofisher.com/content/sfs/brochures/TR0057-Read-std-curves.pdf

https://planetcalc.com/8735/

I have a graph and some tabular format data from BSA experiment. My problém is how would i know the absorbance from graph to know the value of "X"(protein)?