question for in vitro binding selection - (Sep/21/2005 )

Hi,

I wanted to perform an in vitro binding selection to see what consensus motif is for a particular transcription factor. I searched the web and found lots of in vitro binding selection assay use *P32 to label DNAs. Can I avoid using radioactive reagents? Can I simply use ErBr in gel electrophoretic mobility shift assays (GEMSA) to reconver DNA molecules that interact with protein?

Thanks a lot for your advices:)

-bullfrog-

SYBR Gold staining is much more sensitive than ethidium bromide.

-tfitzwater-

Hi tfitzwater,

Thank you very much for your reply So can I say that somehow SYBR Gold can be used, and it is likely to be comparable with conventional methods?

-bullfrog-

Under optimal conditions, SYBR Gold can be used to detect 25 pg of DNA, exceeding the sensitivity of silver staining or radioactivity. (R. S. Tuma et al., 1999. Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators. Analytical Biochem. 268: 278-288.)
Ethidium bromide can only detect 350 pg of dsDNA without destaining or 100 pg with destaining. (H. W. White, N. B. Vartak, T. G. Burland, F. P. Curtis and N. Kusukawa, 1999. GelStar nucleic acid gel stain: high sensitivity detection in gels. BioTechn. 26(5): 984-988.)

Having said that, when we perform aptamer:protein binding curves in acrylamide gels, we use 32P.

-tfitzwater-