Problems with densitometry!....for WESTERN BLOTS

T.S. Pillay tpillay at ucsd.edu
Sat Mar 25 11:50:47 EST 1995


In article <rawlings-2403951039450001 at node138.silcom.com> Jeff Rawlings,
rawlings at silcom.com writes:
>I'm not sure if the chemiluminescent systems are linear (they might be - I
>just don't know). The solution I used was to use I-125 labeled
>secondaries, and develop the blots in a phosphorimager. Both of those
>steps should be linear and quantitative.

Also, when you develop the blot you will find there is a lag of maybe
5-10 minutes before the product builds up - hence if you expose too early
your band will be faint and if you wait a little your bands will be
darker when exposed for the same time at a later stage- this may give the
impression that a shorter exposure produces darker bands whereas in fact
the reaction has not plateaud yet.   This tends to happen when people
develop their blots in the darkroom (not necessary) and start exposing
too early.  I have advised people in my lab to develop the blots on the
bench and then in the time it takes for the blot to be drained and
wrapped and for the trip to the darkroom sufficient time has elapsed for
the reaction to peak.  Does anyone else have a similar experience?



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