Protein quantification - (Aug/04/2013 )
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:
10 = 0.21
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
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.
thank you. Is it not correct? could you please explain it in detail?
What were your OD values of your samples?
Here it is:
Sample 1= 0.28, sample 2 = 0.14, sample 3= 0.37