Amanda Nouwens anouwens at RNA.BIO.MQ.EDU.AU
Fri Jan 15 19:45:52 EST 1999

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. 



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

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