Problems in CHIP assay? - How can i get rid of multiple bands in CHIP assay? (Jul/20/2005 )
Hi all,
In CHIP assay,
1. I am getting multiple bands even at higher anneling temp. Whether I am pulling down junk DNA during the process?
2. I am getting very weak band in Input. How much amount of the sheared chromatin (sample) should be used for Input?
3. I am pulling down protein-DNA complex with primary antibody only. Is it necessary to add secondary antibody?
Thank you for your help
Vaibhao
In CHIP assay,
1. I am getting multiple bands even at higher anneling temp. Whether I am pulling down junk DNA during the process?
2. I am getting very weak band in Input. How much amount of the sheared chromatin (sample) should be used for Input?
3. I am pulling down protein-DNA complex with primary antibody only. Is it necessary to add secondary antibody?
Thank you for your help
Vaibhao
if possible,
1.replace new primers.
2.2 mg protein as strating point.
3.no
Hi, I also have a couple of suggestions to add to zy101
1. Blast primers to genome, it is most important that other sequences do not match to the 3' end of your primers, use the "search for short nearly exact matches" application at NCBI Blast. I had the same problem with my primers, it turned out the 3' end was within a conserved transcription factor binding site which is what gave me the background bands.
2. I use dilutions of the input in order to quantitate the yield amplifying both 0.1%, and 1%. I usually get between 0.5% and 1% pulled down with my IP.
Hi, before you change your primers, you may increase the annealing temperature a little bit and see whether it works.