detect splice variants in living cell

[Tom Anderson]

On Mon, 14 Jul 2008, Peter Ellis wrote:

> On Sun, 13 Jul 2008, DK wrote:
> >>"Zhong Silin" <stxsz1 from nottingham.ac.uk> wrote:
> >>
> >> How can we detect alternative splicing in a living cell?
> >>
> >> I assume that one way is to insert a fluorescent/epitope tag to a specific
> >> region of that protein, which is present/absent in its splice variants.
> >>
> >> Any suggestion please?
> >
> > Why not Northern?
>
> A)  Because he wants to look at protein levels, not RNA levels
>
> B)  Because he wants to do it in living cells
>
> I don't see the necessity for incorporating a reporter tag into the gene
> - would it not be simpler to raise an antibody specifically to the
> variant splice isoform?

Raising antibodies is not exactly trivial. Particularly with that degree
of specificity - if the alternation includes or excludes a big chunk of
protein, you just make an antibody against that, but if it's just a few
residues, you're in trouble.

> Introducing a tag would seem to run a large risk of altering the
> function of the tagged exon.

Yes. This might not matter, though. It might also affect the splicing
itself, which would matter.

Not that i have a better solution!

A particular challenge is looking at a protein with multiple alternative
splicing sites. I was looking at tropomyosin a couple of years ago: four
genes, each with splicing at two sites (or three? i forget). I wanted to
know (dead cells would be fine, in my case), how the pools of A1a, A1b,
A2a, A2b, B1a, B1b, etc compared. I never came up with a good solution.

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk