Just some general questions for all those involved in B-gal immunoassays. I am
currently detecting Western blots on nitrocellulose using a secondary Ab conjugated to
B-galactosidase and X-Gal as the substrate.
1) I am experiencing long incubation times in order to generate signals, ie
>16 hrs @ 37C. I know this enzyme is large (tetramer mw 520 kDa) and has a
low turnover rate (400 molecules/sec) but what could be the problem? BTW, I
also ran a control using only bound B-Gal to the membrane and it still
required >1 hr to get a satisfactory signal.
2) The resulting ppt is green instead of the expected indigo blue product.
My substrate soln contains 1.2 mM X-Gal, 3 mM of both potassium ferro and
ferricyanide, and suitable conc. of NaCl and MgCl all in PBS. Anyone have
similar experiences?
3) I am thinking about switching to a chemiluminescent method to bypass
these long incubation procedures but have noticed a scarcity of suppliers
for B-gal chemiluminescent substrates (Tropix and Lumigen are the two I am
aware of). I do know about the advantages of HRP and AP systems,especially
in terms of specificity and activity, but feel that B-gal enables one to
rule out endogenous enymatic activity. So is there a fundamental property of
B-gal that correlates to the limited number of suppliers, and presumably
lower consumer demand, that I should consider?
Thanks in advance
Larry
