Garry P. Nolan
phactor at netcom.com
Sat Jun 11 22:55:22 EST 1994
dh7y at pop.th-darmstadt.de wrote:
: In article <simmenlab.33.2DF78727 at animal.ufl.edu>, simmenlab at animal.ufl.edu says:
: > 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!
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
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