RNA gelshifts
GENE SIMON HUH
huh at wccf.mit.edu
Sat Jun 12 17:13:00 EST 1993
Dear Netters,
I have been carrying out an electrophoretic gelshift assay in order to
identify factors or complexes which bind specifically to a test RNA. Although
the literature on DNA EMSA is quite extensive, that for RNA is less so (from
what I can see). In particular, the conditions for RNA binding and/or gel
composition differ substantially from paper to paper. I was wondering if
anybody could direct me to appropriate references which address the following:
1) choice of gel buffer system (Tris-glycine? TBE?)
2) gel composition (extent of crosslinking, presence of glycerol in gel)
3) the lowest Kd at which RNA-binding can be detected
4) heparin in the binding reaction; does one risk stripping off specific as
well as nonspecific proteins?
5) use of RNase T1 after binding but before electrophoresis to reduce
unspecific binding and smearing
I would especially like to know how many of the above parameters affect
physical phenomena such as the "caging" effect (i.e., prolonging of
RNA-protein complex half-life).
Many thanks in advance,
Gene Huh
huh at wccf.mit.edu
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