PLEASE HELP...RNA/protein binding

Bill Mackinaw LKMN01A at
Mon Mar 2 19:51:30 EST 1998

I am currently running an RNA-protein binding assay using 32P 
radiolabelled RNA (~400 bp) and an unspecified extract/lysate (not 
purified protein).  It is a membrane filtration assay using 100% 
nitrocellulose.  My problems are twofold:  1)  Reproducibility.  Results 
seem to come and go in an almost arbitrary fashion and seldom repeat.  It 
is worth noting that this assay has produced good results in the past and 
a few of them have consistently repeated.  However, it is nowhere near 
consistent enough to believe.  2)  Controls fluctuate (this possibly 
partially due to problem #1), thereby making calculations based on % 
control binding extremely difficult.
Further compounding these problems is the possibility that the filters-
Millipore 100% nitrocellulose in a 96-well format--have bad quality 
control. They do not always wet as swiftly as you would expect 
nitrocellulose to and when they do wet it is in an erratic fashion.
I am at my wits end--I think this assay could work if the bugs could be 
worked out.  I would welcome any and all ideas or suggestions, or even a 
list of RNA-protein binding pitfalls.

                                                        Bill Smith

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