After successful Transfection protein not detectable - (Feb/06/2007 )
Hello everyone, I hope to get some helpful links...
After transfection and generating stable cell line, I tried to detect the protein by using Western blot.
After immunoprecipitation, doing Western: there were tiny bands in both cell lines, transfected as well as control cell lines. The bands were not more articulately in the transfected cells compared to the control cells...
By RT qPCR, I detected the gene of interest 5-fold higher in the transfected cells compared to the control groupe.
Furthermore, there were a few unspecific bands.
What can I do to get better results:
1) stablelizing proteins?
2) where/what could be the fault?
3) Razing unspecific bands?
thank you for any tipps
5-fold higher in stable line than parent seems very weak to me. What type of promote did you use?
You've got a transcription but no translation of your construct. Did you add (Or is there) a Kozak sequence to (in) your protein? Are you detecting your protein by epitope tagging? Is the epitope tag in frame with your protein?
I'd check the Kozak sequence and also the epitope tag, in this case. If you're using pAb to detect your protein, then have to ensure that the polyclonal had been cited somewhere. If it's a new product, there's no way of confirming if it works.
Thank you for your answer. I am using cmv. Previous studies worked fine with this promoter.