ChIP Assay and no clear answer from professor - (Sep/27/2006 )
I am researching ChIP Assays before I complete my own. My professor wants me to use the Upstate Chromatin Immunoprecipitation Assay Kit. At the same time, he wants me to use Protein A/G PLUS-Agarose beads and Protein A/G beads that were used in another protocol from a paper -Mouse GnRH Receptor Gene Expression Is Mediated by the LHX3 Homeodomain Protein by McGillivray et al.
Following the protocol given to me by Upstate, I am unclear as to where I need to insert the PLUS-Agarose beads. I believe that I need to pre-clear with the PLUS beads instead of the Protein A Agarose/Salmon Sperm DNA that is given in the kit (Part B, Step 3).
The same applies to Part B, Step 5 and 6). Do I add the Protein A/B beads in place of the Slurry and follow the protocol as given from there?
I've attached a link to the protocol for anyone that can take a look at it.
If anyone can help this beginner graduate student I would really appreciate it!!!!
Following the protocol given to me by Upstate, I am unclear as to where I need to insert the PLUS-Agarose beads. I believe that I need to pre-clear with the PLUS beads instead of the Protein A Agarose/Salmon Sperm DNA that is given in the kit (Part B, Step 3).
The same applies to Part B, Step 5 and 6). Do I add the Protein A/B beads in place of the Slurry and follow the protocol as given from there?
I've attached a link to the protocol for anyone that can take a look at it.
[attachment=1797:attachment]
If anyone can help this beginner graduate student I would really appreciate it!!!!
I think what you suggest is correct. The only suggestion I might have is that, if you are starting with a small amount of input chromatin (less than 1-2 million cells worth), you might want to use salmon sperm DNA for both the pre-clearing step (step 3) and the IP step (step 5-6) to avoid non-specific binding. If you have little material to start with then the majority of your chromatin is going to end up binding non-specifically and you'll have little left over to bind specifically.
Since you're just starting out on ChIP, if you haven't purchased the kit already, I'd like to suggest you check out our fast ChIP protocol (no kit required) which we published in Nucleic Acids Research (34(1) e2) and Nature Protocols (1(1) p179-185). From crosslinking cells to PCR ready DNA, the protocol takes 5-6 hours vs. 2-3 days. More important is the fact that the protocol takes a lot less labor (and of course is cheaper than a kit). If you are interested, let me know and I'll send you a re-print of the detailed protocol from Nature Protocols.
That would be extremely helpful! Neither my professor nor myself wants to work on this for 3 days!
Can you sent me the reprint of that nature protocol paper?
My email: zhangh68@gmail.com
Thanks!
I am researching ChIP Assays before I complete my own. My professor wants me to use the Upstate Chromatin Immunoprecipitation Assay Kit. At the same time, he wants me to use Protein A/G PLUS-Agarose beads and Protein A/G beads that were used in another protocol from a paper -Mouse GnRH Receptor Gene Expression Is Mediated by the LHX3 Homeodomain Protein by McGillivray et al.
Following the protocol given to me by Upstate, I am unclear as to where I need to insert the PLUS-Agarose beads. I believe that I need to pre-clear with the PLUS beads instead of the Protein A Agarose/Salmon Sperm DNA that is given in the kit (Part B, Step 3).
The same applies to Part B, Step 5 and 6). Do I add the Protein A/B beads in place of the Slurry and follow the protocol as given from there?
I've attached a link to the protocol for anyone that can take a look at it.
[attachment=1797:attachment]
If anyone can help this beginner graduate student I would really appreciate it!!!!
I think what you suggest is correct. The only suggestion I might have is that, if you are starting with a small amount of input chromatin (less than 1-2 million cells worth), you might want to use salmon sperm DNA for both the pre-clearing step (step 3) and the IP step (step 5-6) to avoid non-specific binding. If you have little material to start with then the majority of your chromatin is going to end up binding non-specifically and you'll have little left over to bind specifically.
Since you're just starting out on ChIP, if you haven't purchased the kit already, I'd like to suggest you check out our fast ChIP protocol (no kit required) which we published in Nucleic Acids Research (34(1) e2) and Nature Protocols (1(1) p179-185). From crosslinking cells to PCR ready DNA, the protocol takes 5-6 hours vs. 2-3 days. More important is the fact that the protocol takes a lot less labor (and of course is cheaper than a kit). If you are interested, let me know and I'll send you a re-print of the detailed protocol from Nature Protocols.