protocol for removing carbohydrates from cell membranes

Michael Grove mjg177 at psu.edu
Tue Jun 17 22:15:41 EST 1997


Does anyone know of a method for removing the carbohydrates from cell membrane 
glycoproteins that will leave viable cells at the end of treatment? 
 I think periodate oxidation  is out of the question.  I'm looking for anyone 
who may have tried using N or O glycosidases for this purpose.  It seems that 
most of the literature on the subject deals with protein extracts and not 
whole cells.  I need whole, viable cells for subsequent cytotoxicity assays.

Secondly, can anyone reommend a universal carbohydrate assay (preferably 
colorometric; my fallback position is a gel mobility assay of the membranes 
with and without treatment) that I could use to verify carbohydrate release 
from my cells into the culture medium supernatant?  I've had fairly nice 
preliminary results using the anthrone reagent, but it doesn't seem to work on 
aminosugars, and will thus probably give a lower than expected reading.  
Likewise, some of the other classical tests for reducing sugars might not be 
%100 appropriate.  Does anyone know if the Molisch reaction will work with 
derivatized sugars?


Thanks in advance - Mike  



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