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ChIP-qPCR (or ChIP-seq) problems with magnetic beads??

ChIP ChIP-seq Dynabeads ChIP-qPCR

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#1 windyhawk

windyhawk

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Posted 20 February 2015 - 03:17 PM

I have been encountering numerous failures using magnetic beads (Dynabeads from invitrogen) to perform ChIP-qPCR or ChIP-Seq experiments. The problem is that I cannot see any enrichment in my samples (even the positive controls) vs the negative controls (IgG, Input) and positive regions vs negative regions.  I did a series of troubleshooting but still could not pinpoint where the problem is:

1. When I switched back to the old but classic ChIP protocol which uses salmon sperm DNA coated Sepharose beads, both ChIP-qPCR and ChIP-seq yielded great enrichment. However, when I used the same antibodies but with Dynabeads, I could not see any enrichment.

2. I compared the Sepharose-based protocol and Dynabeads-based protocol side by side and found Dynabeads-protocol could immunoprecipitate 100 to 1000+ fold more DNA than the Sepharose-based protocol, measured by high-sensitivity DNA analyzer. 

3. I tried several different washing conditions for Dynabeads, such as increasing the washing time, switching to more stringent washing buffer (RIPA) etc. I can see that the extended/more stringent washing condition definitely lowered the DNA yield (by 10 to 80 fold) from Dynabeads-ChIP than that before I optimized the washing condition, but there was still no enrichment when I ran qPCR.

4. I tried anti-rabbit conjugated Dynabeads and used the ChIP validated (by both our lab and other labs) rabbit primary Ab to perform the ChIP with extended and stringent washing condition. I still couldn't see enrichment. 

To be more specific, when I used Dynabeads, I coated the beads with primary antibodies in blocking buffer (0.5%BSA in PBS) at 4 degree cold room overnight and then mixed with the sonicated samples for overnight IP at 4 degree.  After that I washed the beads using washing conditions mentioned above and proceeded  to RNA/protein digestion, reverse-crosslinking for 6-18 hours and DNA purification using QiaGen MiniElute kit. 

 

Have you every encountered this problem? It seems to me that many labs have successfully published their studies using Dynabeads-based ChIP protocol. How can I not get it to work? Where could it go wrong? It is also very counter-intuitive that Sepharose beads which are more porous than Dynabeads should generate higher background. But in my hands, it's exactly the opposite! 

 

The reason I need to use magnetic beads as opposed to Sepharose beads is that I will be doing transcription factor ChIP-seq in a special sample that is not easily accessible.  Therefore, there is a cell number limit for me and I need to maximize my DNA yield from ChIP to be able to prep the library for Next-Gen sequencing.  The Sepharose beads just can't give me enough DNA given my limited cell numbers.  The above optimizations and ChIP-seq experiments were either done using cell lines or looking at histone marks which are very abundant. 

 

Any input is very welcomed. Thank you!


Edited by windyhawk, 20 February 2015 - 03:35 PM.






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