Brendan,
as long as yoy're sure that there is no accidentally introduced stop
codon or frameshift, there might be some kind of cryptic intron
resulting in alternate splicing.You simply might change the linker
sequence of yor fusion, e.g. by site directed mutagenesis.
And, what about an N-terminal fusion or using RFP?
Wolfgang
> From: bkr6 at cornell.edu (Brendan)
> Subject: No fluoresence
> Date: 16 Feb 2001 22:22:32 -0000
> Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre
> X-To: fluorpro at net.bio.net> To: fluorpro at hgmp.mrc.ac.uk
> Help!
> I've put GFP onto the C terminus of my protein and expressed it in
> planta. It retains biological activity but I can't detect fluoresence.
> I've done western blots with antibodies to both GFP and to my protein of
> interest. I can't detect anything with the GFP antibodies but detect a
> smaller than expected product with the Ab's to my protein so it looks
> like the protein is truncated. BUT, he sequence of the clone checks out
> and I've successfully used this GFP vector to make other fusion proteins
> so it seems to be specific to y p.o.i.. Anyone seen a phenomenon like
> this? I'm starting to troubleshoot and thought I would start by
> increasing the linker. Any other ideas? Any insights would be much
> appreciated. Thanks!
> Brendan Riely
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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica
Taipei 115
Taiwan R.O.C.
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