I have generated a stably transfected SK-N-SH cell line that expresses Amyolid beta 42 fused to the pre-proncephalin signal peptide under de CMV promoter.
I checked expression though qPCR and they indeed express the fusion protein.
However, I did not manage to see any signal neither on western blot or ELISA. I measured cell culture medium and monolayer homogenates in RIPA and HCl-Guanidine.
Is it possible that the protein is being arrested at a post-transcriptional level? How could it be that the protein is being over expressed and yet being undetectable?
A possible answer is - indeed - that your RNA is espressed but not translated to the protein, maybe via post-transcriptional expression control/degradation of alien RNA. Another possiblity is that your Abs are not working well (e.g. see Baker, M. NATURE | VO L 5 2 1 "Blame it on the antibodies"). Did they worked reliably before?
Morover, you could check generated RNA sequences for possible generation of unwanted termination sequences within the fusion construct, maybe leading to RNA degradation or NMD.
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