Abdmajid, you should use linear calibration curve OD (595 nm) - C (standard protein, for example, BSA) mg/ml ( coefficient of determination R2 ≥ 0.99)

OD= a C+ b

And after you will determine OD unknown sample, you should use obtained curve

Example https://info.gbiosciences.com/blog/bid/164578/bradford-protein-assay-calculation-of-an-unknown-standard

By using standard curve of protein concentration you have to calculate the concentration of protein. Take a OD of your unknown sample and draw a perpendicular on x-axis from straight line passing through origin of standard curve. So you get the exact concentration of protein in your sample.

• You determine the absorbance vs concentration of a standard protein, whose dye-binding is similar to the protein you are interested in. The concentration range should span the concentration you expect from your sample.
• Then you calculate a regression curve through these points. The Bradford assay does not yield straight lines, but curves. A second order parabola (y = a + bx + cx2) is adequate. This can be done conveniently in most scientific graphing packages (SigmaPlot, Origin, gnuplot, SciDAVis; the latter two are open source).
• Then you determine the absorbance of your sample and calculate the x-value that corresponds to this y-value, that is, you use the inverse of the function you calculated in the previous step.

The crux with this procedure is that the colour yield of different proteins is different. For example, BSA has hydrophobic pockets that bind more CBB (active ingredient in Bradford's reagent) than most other proteins. IgG is a much better standard for this type of assay.

You should measure the concentration of the standards (BSA). Then, plot between the conc. and absorbance to get the slope (Y=aX+b).

Y= the absorbance, X= the conc., a= slope, b= constant.

Measure the absorbance for your protein, Y=aX+b, all of them known, except X unknown which is you protein conc.

you will get the conc. ug/mL convert it to mg/mL; then, divide it by molecular weight of the protein, you will get the conc. in Molar.

thank you for your help

I am still confused how to complete the calculation

I did the slandered curve started from 2ug/100 ul H2O , 4 and 6.

the absorbance was 0.212, 0.256 and 0.36.

for the sample the absorbance was

sample A=0.524

B=0.391

C=0.405

D=0.589

and so on

So how can I complete the calculation?

Abdmajid Hwej, you can't use your obtained 3-point calibration curve (y = 0,037x + 0,128), because OD unknown sample is out of linear range. Coefficient of determination (R² = 0,9481 ) is too low, for calibration curve you should use at least 5-6 points ( 2, 4, 8, 12,16 ug/100 ul H2O )

Thank you Antonina for your reply

I did 8ug=0.357

12=0.509

16=0.615

So how i can complete the protein concentration

This is first time for me doing it

If your obtained data

2 ug/100 ul H2O OD=0,212

4ug/100 ul H2O OD= 0,256

8ug/100 ul H2O OD =0,357

12 ug/100 ul H2O OD= 0,509

16 ug/100 ul H2O OD=0,615

Your 5-point calibration curve y = 0,0296x + 0,1414 ( R² = 0,9935)

C( unknown sample A)=(0,524-0,1414)/0,0296= 12,93 ug/100 ul H2O

C( unknown sample B)=(0,391-0,1414)/0,0296= 8,43 ug/100 ul H2O

C( unknown sample C)=(0,405-0,1414)/0,0296= 8,91 ug/100 ul H2O

C( unknown sample D=(0,589-0,1414)/0,0296= 15,12 ug/100 ul H2O

If your blank (buffer) unknown sample = blank standard

Thank you Antonina Kokisheva for your help.

I think you are ding western blot too much.

I did this week but i got some problems. I run the gel to detect endothelial NOS (t eNOS and p eNOS) in different mice genotype (HT, WT and AMPKalpha1 KO), I used anti t eNOS and ant p eNOS as a primary antibodies and Donkey anti mouse green and Donkey anti rabbit red as secondary anti body, and the DSD gel was 15%. The band was not clear I do not know what is the matter.

I am trying to compere between t eNOS and p eNOS in different mice genotype

Any one can help me to solve this matter?