Gel shift with large DNA fragments (>1 kb) - Can I still do EMSA? (May/21/2009 )
I want to demonstrate protein binding on a whole DNA fragment (about 1.4 kb) using EMSA. I know that the labeled DNA is usually not larger than 500 bp. I was wondering if there is anything that can be modified so I can still do EMSA on these big fragments, or is there any alternative that can be used to demonstrate protein binding on big fragments like this?
Thank you!
-ruiN-
ruiN on May 21 2009, 08:49 AM said:
I want to demonstrate protein binding on a whole DNA fragment (about 1.4 kb) using EMSA. I know that the labeled DNA is usually not larger than 500 bp. I was wondering if there is anything that can be modified so I can still do EMSA on these big fragments, or is there any alternative that can be used to demonstrate protein binding on big fragments like this?
Thank you!
Thank you!
Yes you can, I do it all the time using a fragment(s) that are around 1.2kb in length. I run in an agarose gel and stain with EtBr. The trick I have found is really long run times in a long agarose gel. You might have to use some pretty concentrated protein to get a really nice shift with something that large, but it is totally possible. Hope this helps.
-LT Dusty-