To: protein-analysis at net.bio.net
From: Silvano.Schito at am.pnu.com
Subject: (none)
Date sent: 15 Jan 1999 05:19:55 -0800
Dear Silvano,
> I would like to receive informations about the following techniques:
> 1)Iso-electric focusing of glycosilated proteins such as rh-TPO
Assuming you are using IPG strips for the isoelectric focusing, the following is
my suggestion.
Iso-electric focusing (and 2D PAGE) of glycosylated proteins can be a little
more tricky than non-glycosylated proteins. Sometimes it seems difficult to
actually get them into the IPG strip. I would suggest two things - either use a
sample buffer with strong reducing and denaturing conditions - refer to Molloy et
al, 1998 (Electrophoresis 19:837-844) for details on sample solutions.
Alternatively, you could try boiling your protein in SDS-DTT (standard 1D
sample buffer), diluting the protein with the sample buffer used to rehydrate the
IPG strip, and cup loading the protein.
The above is fairly general - I can provide more specific information if required.
Cheers,
Amanda.
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Amanda Nouwens
Australian Proteome Analysis Facility (APAF)
Macquarie University
Sydney, NSW 2109
Tel: 02 9850 6209
Fax: 02 9850 6200
Email: anouwens at proteome.org.au
