protein elution from SDS-PAGE gels
anouwens at RNA.BIO.MQ.EDU.AU
Sun May 16 20:39:22 EST 1999
I am currently working with membrane proteins, and the best method for elution after
after digestion with trypsin is to add (~8 ul) 50% acetonitrile/0.5% TFA,
sonicate for 10 minutes, and then load ~1ul onto the MALDI target plus matrix.
Try washing the gel pieces in 2.5mM Tris-HCl/50% acetonitrile before digestion.
I have no problem with extracting the peptides, even from membrane proteins
with this method.
If you need more info, let me know.
> Hello, i've been attempting to do mass spectroscopy work with a
> membrane protein, and in order to do so we must do an in-gel digestion
> with trypsin, and then a subsequent elution into ammonium bicarbonate.
> However, when we have tried this with protein stained with Coomassie
> blue, we have had extremely low yields with the elution. So we
> suspect that this is a result of the chemistry behind fixing the
> protein in the gel, and so if anyone has any information on the
> chemistry behind this, or any suggestions on improved methods of
> elution from SDS-PAGE gels, it would be much appreciated. Also, I
> should mention that as this is a membrane protein, and therefore
> extrememly hydrophobic, aggregation may be causing a problem with
> elution as well. Thank you. Also, I would appreciate it if any
> responses could be emailed to me as well, as I am not able to
> regularly check this newsgroup.
> -Dodzie Sogah
> sogah at fas.harvard.edu
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