more about X-gal

carlos h. pedemonte pedemonte at Jetson.UH.EDU
Sat Jan 15 11:31:09 EST 1994


        I want to comment about questions raised on the use of x-gal. I am
using the method with HeLa cells as described by promega and appears to
work well. At least, I have some blue. I read some postings in the net
about different methods used, and I want to ask about the fixing procedure.
Like Andres, I use 0.25% glutaraldehyde for 15 min at room temperature.
Martin uses a mix of formaldehyde and glutaraldehyde for 5 min (T?). My
question is: how much critical is the fixing of the cells to detect all the
b-gal produced? Since b-galactosidase is a soluble protein, I believe the
fixing is important both ways to open the cell membrane and cross-link the
enzyme to avoid diffusion. 
        To test an expression system, I use the plasmid containing the
b-galactosidase gene. I got blue staining, but the level of transfection is
lower than I expected. It is possible that the plasmid, or the liposome
transfection have not work properly. However, I wonder whether it is
possible that some b-galactosidase has been washed out of the cell because
they were not fixed properly. 
        Any comments? Thank you. carlos
Carlos H. Pedemonte                     Telephone:  713-743-1211 (office)
Dept. of Pharmacology                               713-743-1228 (lab)
University of Houston                   Fax:        713-743-1229           
      
Houston, TX 77204-5515                  e-mail: pedemonte at jetson.uh.edu




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