DNA Protein pull down protocol for mass spec - (Sep/22/2008 )
Hi!
I want to start DNA affinity binding assay with subsequent analysis by mass spec. My target DNA sequence is 900bp long with biotinylated 5`end. I want to use Strepatvidin Dynabeads from Invitrogen to catch my DNA-Protein complexes.
I have never done this assay before so far. Therefore I´m looking for a protocol, especially for suggestions how to elute proteins from DNA.
Does anybody have any expiriences with this assay?
Thank you very much!
b.
-borbis-
QUOTE (borbis @ Sep 23 2008, 01:47 AM)
Hi!
I want to start DNA affinity binding assay with subsequent analysis by mass spec. My target DNA sequence is 900bp long with biotinylated 5`end. I want to use Strepatvidin Dynabeads from Invitrogen to catch my DNA-Protein complexes.
I have never done this assay before so far. Therefore I´m looking for a protocol, especially for suggestions how to elute proteins from DNA.
Does anybody have any expiriences with this assay?
Thank you very much!
b.
I want to start DNA affinity binding assay with subsequent analysis by mass spec. My target DNA sequence is 900bp long with biotinylated 5`end. I want to use Strepatvidin Dynabeads from Invitrogen to catch my DNA-Protein complexes.
I have never done this assay before so far. Therefore I´m looking for a protocol, especially for suggestions how to elute proteins from DNA.
Does anybody have any expiriences with this assay?
Thank you very much!
b.
the same questions with the upstairs!
-stone757-
Hi
We use the protocol described in Reddy et.al 1998.
http://bloodjournal.hematologylibrary.org/.../full/91/5/1793
We boil the bead-bound proteins in Laemmli buffer before running on SDS PAGE. I think any standard denaturing WB loading buffer can be used.
-Roald-