PAGE of Glycoproteins

John Troyer nestgrp at earthlink.net
Fri May 31 09:09:48 EST 1996


p9001808 at vmsuser.acsu.unsw.edu.au wrote:

>We have been trying to identify a membrane bound glycoprotein from a protozoan
>for some months now. We run the crude membrane extract on SDS PAGE 12% (4% 
>stacking gel) and electroblot to PVDF membrane.
>  By carbohydrate staining we have discovered that the glycoprotein won't
>even go into the stacking gel and further, will not transfer to the membrane.
>We have tried several brands of PVDF to no avail. 
>   We have investigated the possibility that we have not properly solubilized
>the membrane (all samples incubated at 95 degrees C for 6 min 1% SDS 800mM 
>2-b-mercaptoethanol and 100mM 2-b-mercaptoethanol included in the PAGE running
>buffer) but this has not helped. Does anyone have any ideas or suggestions?


Try running a gradient in your separating gel (4% to 12-15%).  This
will help to get your glycoprotein into the gel.  Also omit the
methanol from your transblot buffer.  Methanol can inhibit the
transfer of large mol. weight species and the transfer will work just
fine without it.   Hope this helps

Regards,

John



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