I have been working with TCEP for quite a while now without (apparent) adverse
effect by 2D gel. This is in short what I have been using:
- I use about 5 mM in the sample buffer. It is ALWAYS added just before use,
that is, I make a concentrated solution (500 mM) and add it at 1:100 dilution in
the sample buffer.
- I use also 5 mM in the rehydration buffer. I typically do not alkylate my
proteins for the second dimension, but for keratins, this is advisable (lots of
cysteines!). Use your usual rehydration buffer containing TCEP first (make sure
the pH is ~ 8.5) for 15 min at RT, then exchange buffer with the alkylation
solution containing your favorite alkylating reagent (I use 10 mM 4-vinyl
pyridine (stock solution: 200 mM in dimethylsulfoxide) ; make sure the pH is
8.5-9.0). That should do the trick.
Good luck
Axel
Annick Daniels wrote:
> Hello everybody,
>
> Has someone of you some experience in using TCEP
> (tris-carboxy-ethylphosphine) for 2D-electrophoresis ?
> What concentration do you use in the rehydrationbuffer/equilibrationbuffer.
> Doe you have some protocol ?
> Are there some special things to account for ?
> I'm trying to get a good 2D-separation of keratins; that's why I want to
> try TCEP instead of DTT. Is someone familiar with 2D-electrophoresis of
> keratins. What do you do to get a good result ?
>
> Thanks in advance for answering !
>
> Greetings
>
> Annick
>
> Annick Dani?ls,
> Innogenetics N.V.
> Industriepark Zwijnaarde 7, box 4
> B-9052 Gent
> Belgium
>
> Phone : 0032-9-241.07.77. or 78
> e-mail : annicdan@innogenetics.be
-- --------------------------------------------------------------------------------Axel Ducret, Ph.D. Senior Research Biologist Merck-Frosst Canada Inc. Dept. Biochemistry and Molecular Biology P.O. Box 1005 Pointe-Claire-Dorval PQ H9R 4P8 Canada
tel. + (514) 428-3428 fax + (514) 428-4900