advice on A/G agarose for ChIP - which gives the lowest background (Oct/26/2006 )
Hi,
I use protein A/G agarose plus from Santa Cruz for ChIP experiments and I always get PCR product in no Ab control (I use around 5 million NT2 cells per IP, preclear with 10 ul of A/G agarose, use 2 ug of Anti Sp1 Ab (PEP2), bind chromatin with 10 ul of A/G agarose, wash three times more than it is recomended in Upstate kit (hand made solutions according to the Upstate kit)).
Also I saturate A/G agarose twice with 1ug/ul of BSA and sonicated salmon sperm DNA!
Can anyone suggest what else I can do to reduce background?
If anyone knows for better A or G beads please let me know (oredring in my country is so difficult and time consuming)!
Grooya
-grooya-
QUOTE (grooya @ Oct 26 2006, 04:51 AM)
Hi,
I use protein A/G agarose plus from Santa Cruz for ChIP experiments and I always get PCR product in no Ab control (I use around 5 million NT2 cells per IP, preclear with 10 ul of A/G agarose, use 2 ug of Anti Sp1 Ab (PEP2), bind chromatin with 10 ul of A/G agarose, wash three times more than it is recomended in Upstate kit (hand made solutions according to the Upstate kit)).
Also I saturate A/G agarose twice with 1ug/ul of BSA and sonicated salmon sperm DNA!
Can anyone suggest what else I can do to reduce background?
If anyone knows for better A or G beads please let me know (oredring in my country is so difficult and time consuming)!
Grooya
I use protein A/G agarose plus from Santa Cruz for ChIP experiments and I always get PCR product in no Ab control (I use around 5 million NT2 cells per IP, preclear with 10 ul of A/G agarose, use 2 ug of Anti Sp1 Ab (PEP2), bind chromatin with 10 ul of A/G agarose, wash three times more than it is recomended in Upstate kit (hand made solutions according to the Upstate kit)).
Also I saturate A/G agarose twice with 1ug/ul of BSA and sonicated salmon sperm DNA!
Can anyone suggest what else I can do to reduce background?
If anyone knows for better A or G beads please let me know (oredring in my country is so difficult and time consuming)!
Grooya
Is there any reason you can't report your results as enrichment above background?
These are the only things I could think of to help with background:
1) You could try increasing the salt concentration a little in your high salt wash.
2) One thing we do is clear the chromatin after the antibody incubation. Apparently we get non-specific antibody chromatin aggregates which pellet even at low speed centrifugation. This means they end up in the final sample and are most apparent in the control. We usually spin at top speed for about 10min after the antibody incubation and then transfer the top 90% to the tube wih the beads.
As far as other brands of beads, we use Fast Flow protein A agarose from Amersham. It works great for us but it's vey high capacity and thus may give you more problems with background.
-KPDE-
QUOTE (grooya @ Oct 26 2006, 04:51 AM)
Hi,
I use protein A/G agarose plus from Santa Cruz for ChIP experiments and I always get PCR product in no Ab control (I use around 5 million NT2 cells per IP, preclear with 10 ul of A/G agarose, use 2 ug of Anti Sp1 Ab (PEP2), bind chromatin with 10 ul of A/G agarose, wash three times more than it is recomended in Upstate kit (hand made solutions according to the Upstate kit)).
Also I saturate A/G agarose twice with 1ug/ul of BSA and sonicated salmon sperm DNA!
Can anyone suggest what else I can do to reduce background?
If anyone knows for better A or G beads please let me know (oredring in my country is so difficult and time consuming)!
Grooya
I use protein A/G agarose plus from Santa Cruz for ChIP experiments and I always get PCR product in no Ab control (I use around 5 million NT2 cells per IP, preclear with 10 ul of A/G agarose, use 2 ug of Anti Sp1 Ab (PEP2), bind chromatin with 10 ul of A/G agarose, wash three times more than it is recomended in Upstate kit (hand made solutions according to the Upstate kit)).
Also I saturate A/G agarose twice with 1ug/ul of BSA and sonicated salmon sperm DNA!
Can anyone suggest what else I can do to reduce background?
If anyone knows for better A or G beads please let me know (oredring in my country is so difficult and time consuming)!
Grooya
Here's a few more ideas.
In our experience, a high salt wash had little effect on background but possibly, stopping non-specific binding before it even happens (during antibody binding) by incubating with higher salt during antibody binding (rather than during the wash), this might be more effective.
You could also decrease the concentration of your chromatin during the IP by diluting with buffer. This is actually the most common way we deal with high background. Try a few different concentration till you find one that works. The non-specific binding will decrease faster than your specific binding when you decrease the concentration of the chromatin.
-KPDE-
thank you very much for the ideas!
GROOYA
-grooya-