Hello,
I'm struggling with the unequal beta-actin expression level on western blot. I did the BCA twice and got the similar result, I'm sure the loading is correct. The loading amount should be 25 ug.
There are clearly two groups, and they are equal amount each groups. The first two samples (from the left side) were lysated with CelLytic M and protease cocktail. The rest of sample were lysated by RIPA buffer with a different kind of protease cocktail and PMFS. So I'm thinking this unequal beta-actin is due to the lysis, but didn't find any evidence.
Could anyone help me with this issue or give me some advice on this? Thank you so much.
Dera Yajin Sun,
Until you protein quantification is correct, this is not an isolate case that B actin would vary, check with another menage protein like alpha tubulin.
All the best
Hi Yajing,
RIPA interferes with most of the protein quantification kits mainly due to high detergent concentration. If you add the same amout of RIPA that you use for the BCA to your assay, does it give a colour change?
Try diluting your protein in PBS (1:10-1:100 if the concentration is high enough) or switch to an assay that can be used with detergents. (Expedeon makes BradfordUltra which works very well with RIPA)
I agree with Victoria, sorry i didn't notice the 2 groups was made with 2 different lysis buffer.
Did you try to make a protein coloration ?
@Victoria, Thank you so much.
I didn't think before that RIPA can interferes the protein quantification. Now I believe in it. I will try to use the same lysis buffer with the sample and to do western blot again.
@Sylvie,
Thank you for your advice. I didn't do protein coloration at that time. I will try that on my next experiment.
Dear Yajin
BCA reacts with aromatic amino acids even if the proteins are degradated. Some detergents, like N-lauryl-sarcosyle, have proteases inhibitor activity. It may be that the cell lysis with CelLytic M and protease cocktail is better than Ripa buffer. Load a double volume of the samples 3-4 and 1+1/2 volume of the last sample.
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