b-Gal

[dh7y at pop.th-darmstadt.de]

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


Hi Karen!

Sure I can help you!

1) BioTechniques Vol. 7, No. 6 (1989) pp. 576-579
   Transfected cells were rinsed with PBS and 2 ml HBSS containing 3.5 mM ONPG
   were added to the cells. After incubation at 37 C (2-10h) in the incubator
   the solution was removed and absorbance is read at 420 nm. 
   You can also include 0.5 % NP-40 to the HBSS/ONPG-solution, here you can
   start reading absorbance after 15-90 min.

2) JBC Vol. 269, No. 4 (1994) pp. 2550-2561
   Here is a protocol for handling lots of probes in microtiter plates.
   However cells are lysed in the plate, freeze-thawed and measured directly
   without removing cell-extract.

3) Simply fix cells e.g. with glutaraldehyde and stain cells with X-Gal

But be carefull you don't measure internal eucaryotic beta-galactosidase!
Need help on this?

4) Analytical Biochemistry Vol. 215 (1993) pp. 24-30
   Paper focusing on selective inactivation of eucaryotic beta-galactosidase
   in cell-extracts. Important!


Hope this will help you!

Last but not least: What cells do you transfect with which method? What plasmid
construct do you use? Do you know something about endogen beta-gal activity in
endothelial cells?

Bye, Michael :-)


Michael Teifel
Technical University of Darmstadt
Institute for Biochemistry
Petersenstrasse 22
D-64278 Darmstadt
Germany
Tel. +49-6151-165157
Fax. +49-6151-164171
Internet dh7y at pop.th-darmstadt.de