[Histonet] in situ hybridization histochemistry question about radioisotope

Susan Bachus susanbachus <@t> verizon.net
Sun Mar 14 01:28:12 CST 2010

I have routinely seen that we obtain slightly different results (like 10-20% 
variance) in using 35S dATP for TdT-catalyzed tailing reactions of 
oligonucleotides from month to month as we use different batches (made fresh 
monthly) of the 35S dATP.   But on a few occasions over the years we have 
found dramatically reduced results with a particular lot.  This happened 
again in February, and I confirmed by comparing directly between the Feb. & 
March lots in March that the results with the Feb. lot were half that of the 
March batch (of course, given the half-life of 35S, they would be expected 
to be down some, but not by half!).  The company was good about believing me 
and not charging us for the March "replacement" batch, but they have no idea 
what could cause this to happen occasionally.  They check the specific 
activity, pH, purity, etc. and run a "bio-assay" that is a binding assay 
each month, but were not set up to test the enzymatic reaction.   I told 
them that years ago when this happened it was finally determined that the 
DTT concentration was unusually high and interfered with the reaction, but 
they discounted this possibility.  I guess I should just count my blessings 
that things are working again now (mercifully, I was able to cling to 
optimism, based on past experience, that the isotope was the problem, until 
I could confirm this myself), but it's very frustrating that the company is 
not motivated to check the efficacy of the item for this particular 
application (which I would imagine a lot of customers use it for) each 
month, or to figure out what causes this to happen on occasion (so as to 
avoid it!)--we lose a lot of time and other expensive reagents (e.g. the 
TdT) each time this happens.   I also don't know how to interpret it--I know 
that as the enzyme begins to degrade it just means that the tails are 
shorter, which I can compensate for with a longer film exposure, but I have 
no idea what's going on when the isotope causes the problem & am afraid to 
use those batches.  Does anyone else using ISH have any ideas about what 
causes this variability?   grateful for any thoughts,   Susan 
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