advice in western blotting - (Mar/17/2014 )
I need ur advice in protein quantification for western blotting.
I have tried to quantify my Proteins obtained from cell lysate before start my western experiment,
I prepared 20, 10, 5, 1, 0.5, 0.1, 0.05, per 10 ml of Bovine serum albumin solution.
the I used rapid CCB (nacalia tesque solution for protein quantification).
I did not get linear relationship, but the best fitting line I got from log conc vs absorbance as indicated in the attached STD curve.
I got correlation coefficient =0.97.
According to such STD curve, conc of Control is 3 times conc of test so I had to make 1:3 dilution.
and I got significant results when u compare test to control.
but I am skeptical about my protein concentration calculations for several reasons:
1) I am not convinced of absorbance results for each conc separately.
20 mg conc #abs =1.3
10 mg conc #abs = 1.25
5 mg conc #abs = 1.21
1mg conc #abs = 0.85
0.5 mg conc #abs = 0.65
0.1 mg conc #abs =0.38
0.05 mg conc #abs = 0.34
Control # abs = 1.76
Test # abs = 1.56
for example 10 mg #abs should be 10 times 1 mg # abs, but it is not the case, and it is not the case in each absorbance results for each dilution.
Please check my attached STD curve
2) I feel like I have some precipitation when I added my samples (Protein from cell lysate) to comassai dye (Rapid protein quantification solution).
Can u give me ur suggestion regarding such STD curve.
if u have good suggestion regarding protein quantification
coomassie based protein assays are not linear. there is a relatively small range of protein concentration where there is a semblance of linearity. 2 mg/ml (20 mg/10 ml) is beyond the "linear" portion.
also, albumin as standard gives ~2x the response of many other proteins. we routinely us a gamma globulin standard.
here is a publication from biorad that may help.