Electroelution and deglycosylation

Prasad, Krishna Prasad at biologysx.lan.nrc.ca
Sun Oct 9 17:51:36 EST 1994


This is in reply to the query from Yueqiao Huang:
                                                              
(yqhuang at iastate.edu ()
Subject: Electroelution and deglycosylation

> I want to deglycosylate a protein purified by immunoaffinity
> chromatography. Since  the monoclonal antibody recognizes three bands on
> the Westernern, and I am only interested in one of them, I will have to
> separate that band from the others after immunoaffinity column and
> SDS-PAGE.

> I tried to cut the band on the PAGE gel after aqueous Coomassie Blue
> Briliant R-250 staining, and electroeluted the band by using IBI
> electroelutor. But I recovered almost nothing.

> My questions are:

>. Can I transfer the bands to a nitrocellulose or PVDF membrane, stain
>he bands, cut the band of interest, and electroelute or extract (by
>ther means) the protein? If yes, how?

>. Can I just do deglycosylation on Nitrocellulose or PVDF membrane and
>hen analyse the deglycosylated core protein? any refenences?

>. If I have to electroelute the protein either from the membrane or from
>he gel, what are the good device you have been using? BioRad? Hoeffer?
>r any other? 

>ny hints/tips/suggestions would be greatly appreciated.

> Yueqiao Huang

-------------------------------------------------------------------
I have the following reference for you on carbohydrate analysis performed 
directly on PVDF western blotted protein bands:

M. Weitzhandler et al., J. Biol. Chem, (1993) 268, 5121.

Hope it helps and works.

Good luck.

                                           #########
Krishna Prasad                             #########
Institute for Biological Sciences          | @   @ |
National Research Council Canada          {|       |}
Ottawa, Canada                             | \___/ |
                                           |       |
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~--------~~~~~~~~~~~~~~~~~~~~~~~~
"Some are born Great                          |  |
 Some achieve Greatness and                  / >< \
 Some have Greatness thrust upon them"     /|      |\

 (From Shakespeare's works, if I remeber correctly)




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