advice in western blotting - (Mar/17/2014 )

Dear all,

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.

for example

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

another thing 

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

Madelin

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.