PLEASE HELP...RNA/protein binding
LKMN01A at prodigy.com
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 LKMN01A at prodigy.com
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