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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