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immunolocalization q's

Martin Latterich micro at mendel.Berkeley.EDU
Thu Aug 18 17:58:10 EST 1994

In article <330o7a$rd5 at news.umbc.edu>
bolognes at umbc.edu (Ms. Cynthia Bolognese; (BIOL; GRAD)) writes:

> I'm trying immunofluorescence with yeast, and the major problem I have 
> encountered is that my cells don't stick to the slide! I have been coating
> the slide with 1mg/ml poly-L-lysine from sigma (supplied as a solution).
> The cells stick all the way until it's time to add the secondary antibody. 
> Does anybody have any solutions or references? Thanks in advance!

Have you tried SDS denaturing spheroplasts for short amounts of time? 
The negative charge conferred by SDS will bind spheroplasts tighter to
the poly-lysine coated slide. The concentration and time of SDS
treatment may depend on your antibody.  A good reference is Roberts et
al. (1989) JCB 108, 1363-1373 and Pringle et al. (1989) Meth. Cell
Biology 31:357-435.

Good luck,


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