Re counting prot bands after SDS PAGE

leon harris harris at possum.murdoch.edu.au
Mon Jan 31 04:00:42 EST 1994


D Fitzgerald asked for a method for counting radioactive 
bands after SDS PAGE. RG Waters suggested using a digest 
with H2O2 and conc ammonia. You certainly can do this, 
but there is a very nasty pitfall for the inexperienced. 
You get a massive amount of chemoluminescence which can 
artifactually give you hugely inaccurate DPM. There are 
a couple of solutions to this: 1) let the digest stand 
for a few days until the chemoluminescence dies down; 
2) if 
you have a Packard counter (sorry no model # but after 
the early 80s) you can set the energy window so that the 
first 4 Kev's are not counted (chemoluminescence causes 
lots apparent low energy "dpm"); or 3) I seem to recall 
that you can add glacial acetic acid (200 ul per 
5ml) to the mixture after digestion and this eliminates 
chemoluminescence.  Incidently, if you have a Beckman ls 
series counter, the rcm column stands for random 
coincidence monitor (ie chemoluminescence: I don't know 
details for other machines).

Best wishes all,

Leon
Harris at possum.murdoch.edu.au






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