Cell surface Biotinylation

[Gary Rudnick]

In article <ANITA.94Mar14111140 at piccolo.cco.caltech.edu> anita at piccolo.cco.caltech.edu (Anita Gould) writes:
>From: anita at piccolo.cco.caltech.edu (Anita Gould)
>Subject: Re: Cell surface Biotinylation
>Date: 14 Mar 1994 19:11:38 GMT

>   From: rtfm at cix.compulink.co.uk ("Peter Smith")
>   Organization: Abort, Retry, Fail Ltd
>   Date: Sat, 12 Mar 1994 21:39:04 GMT

>   Hi Folks, 
>           Does anybody out there have a good method for labelling cell 
>   surface molecules with biotin? I've been using 125I labelling with pretty 
>   good results, but my finger tips are getting fried with all that 
>   radiation!  I'm sure that labelling the cell surface with biotin is 
>   possible....all I need is someone to tell me how!! Also any useful tips 
>   or pitfalls would be appreciated, I want to immunoprecipitate from the 
>   cells once they are labelled, will this be a problem?
>           Thanks in advance from me and my finger tips!

>   Theresa

>Yes -- some folks in my lab developed & published a biotinylation
>method.  The ref is:

>Kayyem, JF et al, 1992. A method for the generation of monoclonal
>antibodies against rare cell-surface molecules.  Eur J Biochem
>208:1-8. 

>The only erratum/caveat I know of is that somewhere in the paper it
>mentions a step done at 4 deg, but it was actually done at 16 deg,
>because there's SDS in the buffer, & it ppts at 4 deg.  (I'm pretty
>sure it was the avidin column step, which it sounds like you're not
>necessarily even going to be doing, unless you're going to go on to
>purify the cell-surface molecules).

>If you need any help or any further info, just drop me a line, & I can
>give you phone/fax/email addresses for people here to talk to.  

>                                Best of luck!

>--

>                                   -Anita Gould
>                                    anita at cco.caltech.edu

Here are two references that you could use for techniques:

1. Pietrini, G., Y. Suh, L. Edelmann, G. Rudnick and J. Caplan.  
The axonal GABA transporter is sorted to the apical membrane of polarized 
epithelial cells.  
J. Biol Chem.  269:4668-4674 (1994).  

2. LEVYTOLEDANO, R., L. CARO, N. HINDMAN and S. TAYLOR.  
STREPTAVIDIN BLOTTING - A SENSITIVE TECHNIQUE TO STUDY CELL SURFACE  PROTEINS 
- APPLICATION TO INVESTIGATE AUTOPHOSPHORYLATION AND  ENDOCYTOSIS OF 
BIOTIN-LABELED INSULIN RECEPTORS.  
ENDOCRINOLOGY.  133:1803-1808 (1993).  



================================================================
Gary Rudnick                       |voice (203) 785-4548
Department of Pharmacology         |fax (203) 785-7670
Yale University School of Medicine |bitnet RUDNICK at YALEMED
333 Cedar Street, P.O. Box 208066  |internet:
New Haven, CT 06520-8066           |RUDNICK at BIOMED.MED.YALE.EDU
----------------------------------------------------------------
What separates the men from the boys is the price of their toys.