b-Gal

[Garry P. Nolan]

dh7y at pop.th-darmstadt.de wrote:
: 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  :    
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!

Karen,

I'll add to this that you can also use methyl-umbelliferone-galactoside 
(MUG) added directly to cells.  Gets into cells readily and the product 
equilibrates rapidly.  Just add it to approximately 1 mM.  test by 
fluorimeter UV excitation, blue emission.  Available from sigma and 
excitation/emmision wavelengths in Molecular Probes.

Garry P. Nolan
Molecular Pharmacology
Stanford University