bradford assay calculation

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Anukumar
Anukumar's picture
bradford assay calculation

Hi all. I have to do SDS-PAGE. i have done bradford assay to know protein concentration of my sample protein. I use BSA as standard 1 mg/ml. Prepared strandard curve (absorbance value that are subtracted from blank). I want to know that every time when i will plot standard curve should we take 0 reading on x and y axis. 2) Should we have to subtract the blank absorbance value from our sample protein absorbance also. 3) I want to know the concept of dilution factor multiplication with the protein concentration. For example if take 10ul of sample volume and 90 ul water, then what should be the D.F. is it 10 or 90. 4) I added 1ml bradford in 10ul of sample+ 90ul water mixture, so during calculation should i have to divide the protein concentration in ug/ml with 1000ul of bradford volume.

Aaditi Lele
Aaditi Lele's picture
Bradford Calculations

If you are taking 10ul of sample and 90ul water , then the dilution factor is 10 fold. Think of it as you are taking 1 part of the sample and adding it to 9 parts of the buffer making a total of 10; so you are diluting it 10 times. Hence the dilution factor is 10. For your other questions, refer to this web page. It has the detailed information on all the calculations and methods employed for Bradford:https://info.gbiosciences.com/blog/bid/164578/bradford-protein-assay-calculation-of-an-unknown-standard Thanks !