Membrane proteins

[TCruz from ibmc.up.pt]

   Dear all,


   I work with membrane proteins of Leishmania and I have b= een having
   some problems.


   First= , does anyone have experience with chimeras? I did a GFP and a
   c-cmyc fusio= n protein and none of them could be detected by western
   blot. Both fusions = are C-terminal, though I also did two N-terminal
   c-myc tagged proteins and = again nothing was detected.


   = Sec= ond, I tried to produce antibodies against those membrane
   proteins using re= combinant polypeptide and although it recognizes
   the recombinant truncated = protein, it does not recognize in parasite
   total extracts (in SDS-PAGE). Is= this common?

   
   = Due to this result= I introduced an episomic copy of the gene in
   order to over-express the pro= teins in the parasites. Since I could
   not detect the proteins by western bl= ot, I went back to qPCR trying
   to understand if there were at least higher = levels of mRNA. Indeed I
   saw a fold increase of around 70 for one of the pr= oteins and 4 for
   another one. The other 2 proteins did not show any increas= e in the
   mRNA levels.

   So, in conclusion, I wonder if the parasites could be able to eli   minate the mRNA so fast that no protein is detected and how come the
   parasi= tes don't have any mRNA if they carry an episomic copy of the
   gene in= serted in an expression vector.


   I know this is a lot, but can you help me?
   I thank you all in advance,


   

   Tânia Cruz

   Iron Genes and Immune System
   Institute for Molecular a= nd Cell Biology
   Rua do Campo Alegre, 823
   4150-180 Porto
   PortugalPhone: +351-226074956
   email: [1]tcruz from ibmc.up.pt
   
References

   1. 3D"mailto:tcruz from ibmc.up.pt"