help! Truncated protein cannot expressed in mammalian cells?
Posted 09 March 2009 - 09:53 AM
Posted 09 March 2009 - 10:42 AM
Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley
Posted 09 March 2009 - 10:59 AM
I assume these are denaturing gels? You say that you cannot detect the truncated fusion proteins with the myc antibody. Can you detect them with your protein target antibody still or does that also disappear? If both have disappeared, have you tried producing them by in vitro transcription/translation reactions? Sometimes loss of a protein region or even a few bp changes significantly alters recombinant protein expression, so, perhaps your expression is merely lower than usual. You could try immunopurifying your protein with myc antibodies and protein G beads before loading onto SDS-PAGE to concentrate it. Also, make sure that your recombinant proteins don't have any codon usage problems due to the new fusion sequence.
Dr Teeth, thanks for your reply. My target antibody only detect the c-terminal part of the protein, which means it won't recognize the truncated protein when I delet the c-terminal. For one of the constructs I retained the c-terminal, that antibody couldn't work either. The thing is wether I delet the N or C or even a middle part of the protein, I cannot detect anything. Even when I pull-down with protein G, it didn't show anything. BTW, I fused the proetin fragments with a GFP N-terminal tag as well, it seems the expression of GFP-fused proteins have no problem. I guess I will try the in vitro transcription, but in that way, I cannot do my following experiments.
Posted 21 April 2009 - 02:23 PM
Posted 22 April 2009 - 09:35 AM
This was my thought as well. You may be getting expression but the mutant is being degraded just as quickly and hence, no detection. Try treating your cells with a proteosome inhibitor and see if you can detect the protein then.
May be the proteins were unstable due to truncation and degraded. Are you sure the truncated proteins are expressing well??
Posted 24 April 2009 - 08:43 PM
Understanding Life One Protein at a Time...
Understanding Life One Protein At a Time...
Posted 16 November 2012 - 05:42 AM
please send me an email at hegyihedi AT gmail.com