Protein quantification - (Aug/04/2013 )

Hello

I just performed bradford assay. My BSA stock is 1mg/ml. I did standard curve using 10, 20, 30, 40 and 50 ug (1mg/ml BSA stock). These are the values:

ug 0.D

10 = 0.21

20= 0.42

30= 0.69

40= 0.89

50 =1.05

Y = 0.0215x+0.007 and R2 = 0.9926

I extracted my protein and dissolved in 500 microlitres of IEF buffer: After I did my bradford assay, I got the following values:

ug/10 ul = sample 1) 12.69767, sample 2) 6.186047, sample 3) 16.88372

ug/ul = sample 1)1.26, sample 2) 0.61, sample 3) 1.68

Does it mean my 1st sample has 1.26 mg/ml? I have doubt because I dissolved in 500 microlitres of IEF buffer to dissolve my pellet (from this I took 10ul) and to store at -20C

I also need same amount (600ug) I can add upto 300-350 ul for for my IPG strips. But I got different values, Is there any formulae to calculate

Could you please answer to the above query

Thank you

-chandch-

The buffer you are using can influence the bradford. Make sure that your blank and standard curve have the same final concentration of all buffer components as your lysate.

-doxorubicin-

thank you. Is it not correct? could you please explain it in detail?

-chandch-

What were your OD values of your samples?

-Missle-

Here it is:

Sample 1= 0.28, sample 2 = 0.14, sample 3= 0.37

Thank you

-chandch-