SOKATCH.BURNS@BIONET-20.BIO.NET (P. Gayle Burns) (04/17/89)
Thanks! Yes, the Promega GRAB kit was the one I was thinking of. The original method on which this kit is based is from Levens and Howley, Mol. Cell Biol. Vol 5, 2307-2315 (1985). Also see Gimble, Levens, and Max, Mol Cell Biol Vol. 7, 1815-1822 (1987). Another affinity method that was brought to my attention is this: The DNA fragment is affinity labeled with biotin-deoxynucleotide by filling in the end using Klenow. This binds to streptavidin-agarose (BRL) and you have your affinity matrix! After binding of the repressor from a crude cell lysate, the protein can be eluded by high salt concentration, inducer, or as a complex with the bound DNA fragment by restriction digestion. The authors claim a 3400-fold enrichment. This method is by Leblond-Francillard, Dreyfus and Rougeon, Eur. J. Biochem vol 166, 351-355 (1987). I'd like to hear of successes and failures with these methods. Gayle Burns -------