Detecting b-gal in cultured cells ?

Dave Bates dobates at ucdavis.edu
Fri Jun 10 14:33:04 EST 1994


In article <simmenlab.33.2DF78727 at animal.ufl.edu>
simmenlab at animal.ufl.edu writes:

> Hello:
>                 
>               I am transfecting cells in culture with a b-gal expression 
> vector.  I was wondering if it was possible to detect b-gal expression without 
> preparing cell extracts.  I just want to know if the transfection worked or 
> did not work.  If anyone has any experience with this please let me know.  
> Thank you very much!
> 
> 
>                                                           Karen Reed

For cultured endothelial cells I wash with PBS,  fix with
2%Formaldehyde, 0.1%Glut, Wash again and stain O/N at 37C with X-Gal
assay buffer (60uls 50mg/ml X-Gal, 75ul each of  K Ferro and
Ferricyanide (0.2M), 12uls MgCl2 (1M), made up to 3mls with PBS).
Worksd a treat. Can also use anti B gal antibodies (from lots of places
eg Sigma), or for in vivo work without fixation, Image green or Imagene
Red from Molecular Probes.

Good Luck
----------------------------------------------------------------------
Dave Bates,                                        dobates at ucdavis.edu
Department of Human Physiology, University of California at Davis
 
No I don't have a sense of humour, now buy me a beer
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