In WB the protein level was lower in the overexpression cell line
My antibody is against human ,rat and mouse
I did an overexpression of my protine using human cDNA in a normal mouse mammary cell line, I was able to detect the overexpression by qpcr but not by Western blot. Is there any idy why I couldn't detect it ?
Hi Faris
What proteins are you trying to detect? Are they from cell lysates or secreted proteins (from cultured media)? You have to retrace your steps and double check if you used the right protocol for protein extraction. Are you working with a fresh set of antibodies or have they been used by somebody else who has proven that they work?
Then again, most experiments dealing with gene regulation does not necessarily translate to the expected production of the proteins in question. This is why the clinical significance of a research dealing with the functional effects on genes more often than not, involve western blots to check/confirm if the effect on the mRNA leads to the expected protein production. (upregulation of gene = more protein translated, downregulation of gene = less protein translated).
Wishing you the best of luck!
Hi Faris.
Can be for many reasons. Since your antibody seems to work, it is more liekly that expression does not.
I would double check the expression cassette first (promoter, Kozak sequence, everything is in frame, poly A signal near the stop codon. As Sepide pointed out, there are also posttranscriptional issues.
Using a tagged form of your protein for transfection (GFP or flag or...) may also be an option.
Good luck
best
chrstian
Dear Faris,
I want to ask few questions,
1) Did you clone the cDNA into the plasmid by yourself?(then follow Christian's answer) PCR will be positive even the cDNA is in non-coding frame in the plasmid.
2) How did you transfect? what about transfection efficiency?Many times due to less transfection efficiency we can not observe the overexpression, if both endogenous and overexpression have same molecular size.
3) At what time point did you extract the protein?(I wish you would have done after 24hrs of transfection) Level of expression varies from time to time depending on our protein stability.
Note: If you have any doubts do not hesitate to ask and be more informative in your questions.
Thank you
I didn't do the transfection by my self.
The cells that overexposed the gene behaves different from the original one and I can see all the biological effects that was expected though the problem in WB detection , is it possible that the overexertion gene is more amenable to degraded than the endogenous one?
- Badges
- Science method