Dear Samer, that is quite an important point for construct design especially in the context of signal sequence. That is not a trial task, but based on the information you have shared with us I would assume that your protein does not get properly processed in the ER if you cannot detect it by Flow cytometry (assuming you Ab against the HA tag works properly). If you are detecting it on WB, the question is that is fit the expected MW or is it smaller sized protein? Is it possible to be an unspecific band? The tag should not be cleaved as the cleavage should happen between the SS and your HA/Myc tag. Also normally the SS will be cleaved co-transcriptionally therefore if you can detect it via WB it is already confirmation that the tag is there. It is mostly you are disturbing the post-translation folding/targeting which keeps your protein in the ER and therefore you might detect it via WB on the WCL, but not on Flow cytometry as it is not represented on the surface.
I hope that helps, Best wishes Nikolay Dobrev On Thu, Jun 22, 2023 at 11:57 AM samer halabi <000030c2162795b2-dmarc-requ...@jiscmail.ac.uk> wrote: > > Dear All, > > Sorry to be disturbing you all with my inquiry. > > I am tagging a membrane protein (MHC I) just after the Signal peptide with > either HA-tag or Myc-tag and expressing it in mammalian cells. I am able to > detect that protein by immunoprecipitation followed by Western blotting using > anti-Myc antibody or anti-HA antibody. However, I could not detect it by > flowcytometry, which could mean the tag was cleaved off or the protein is > incapable to be delivered to the cell surface among other possibilities . > > Does anyone has any experience with tagging membrane proteins after the > Signal peptide? Is there any restrictions against that? Or a better tag to > use? > > Thank you. > Best regards, > Samer > > ________________________________ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
