How can I extract Chromatin-protein complex without using Chip? - The cheapest method to prove a protein to bind DNA (Jul/26/2006 )
Does anyone have experience to check whether a novo protein can bind on chromatin without knowing the specific regio of chromatin that it can bind?
As I know doing Chip require to know the suspected specific region of chromatin that a protein would or may bind. After immunoprecipitation, one can amplify this suspected region with PCR. BUT...... I have no idea where my novo protein would bind. Can I just isolate DNA-Chromatin complex with chemistry method? Do you have protocols?
Another question, do anyone know about the history that how people know histones bind the chromatin?
One simple approach is to lyse cells in a 1% NP-40 buffer (50mM TrisHCl, 150mM NaCl, 1% NP-40) and spin, say 5 minutes maximum rpm in a microfuge. In theory the pellet should contain the cellular DNA and chromatin bound proteins. Resuspend the pellet in an SDS sample buffer, boil and your set for Western blot analysis.
I'm little confued about your method~
Lysing cells in the weaken cell lysis buffer can preserve the protein-dna interaction?
how can we know if the protein can bind dna from SDS-PAGE?
Thanks for the answer first
if you have an antibody for your protein you could look at the subcellular localisation of your protein of interest, if it's nuclear, then it's hghly likely to bind to DNA, if you then go an look at metaphase chromosomes then it should paint, or light up along the arms of the chromosome if it binds to chromatin.