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?
>> David Cross
> University of NSW Australia
>p9001808 at vmsuser.acsu.UNSW.edu.au
How about a phenolic group that is binding? Try urea as h-bond
breaker?
JB
Other ideas: try some acetone in water-buffer.
