We bought Dynabeads from Invitrogen attached to protein A to perform ChIP assays. The reason is that apparently they were easier to work with than the agarose ones and suitable for sequencing as they were not blocked with salmon sperm.
I am having lots of problems with them when I get to the washing steps to remove the inespecific binding: the last buffer which contains LiCl at 250mM coagulates in the tube with the chromatin, antibody and Dynabeads after 5 minutes of rotation. It turns into a thick gel, making it impossible for the beads to travel across it and attach to the magnet.
I found out later that probably keeping the wash buffers on ice could have triggered this incident. Surprisingly enough, when I made a try with different amounts of chromatin only the tube with the greatest amount of chromatin turned shortly into a gel when kept on ice. Therefore, the amount of chromatin used may be as well somewhat relevant.
I have been using the wash buffers at RT lately, but I still have trouble with the beads because even though the buffer doesn´t coagulate so firmly as before, not all of them would attach to the magnet and many are lost in the process. Additionally, if the tube is heated up a bit the gel becomes liquid again, but I don´t think I should heat up my sample to avoid the coagulation, otherwise I might ruin the chromatin.
Does anybody knows wether the Dynabeads could be denatured somehow with a buffer containing LiCl at that concentration or what could be the problem here? I have seen in other protocols that higher concentrations of LiCl are used for washing these beads, so that would be unlikely...
Thanks in advance!
what is exactly the composition of your buffer? normally if there is no detergent in there (for instance Na-deoxycholate, Triton or NP40) this tends to happen to the dynabeads..
Thank you for your suggestion, but this is not the case: the buffer contains Tris-HCl, Na-deoxycholate 1%, EDTA, NP-40 0,1% nad LiCl 250mM
is your sample size is more than what it should be,
once reduse sample size and try it,
is RNA problem, becose Licl precipitate RNA very well, and may be avery thing precipitate with that.
Thank you Praveen for your suggestion. I believe that the sample size is adequate, but the issue with RNA is something I never thought that could be a problem. Yes, I guess it could be a factor interfering with my assay, thank you.
Hi!
Do you have a negative ct? E.g beads w/o antibodies or nonspecific antibody? This is an important control anyways. If so, do you see the same with the neg ct?
Alternatively, it could be too much chromatin. How do you quantify chromatin and how much do you use?
Have you kept going untill the final and had a look at the qPCR results?
Finally, I dont think you should ever heat the chromatin.. Except at the decrosslinking step of course :)
Vegard
Hi Vegard,
We use 12,5million cells per tube to sonicate and then we split that in two IPs.
In fact we don´t quantify the "raw" chromatin as the measurement from the nanodrop doesn´t seem very reliable: in chromatin DNA and proteins are present at the same time and each one could be considered as contamination in either a DNA or protein assay. What we do is quantify the DNA purified from the input.
Yes, I could get till the PCR step in a couple of occasions, but the result was not very convincing because:
-In one PCR two out of eight genes resulted well (i.e had a big fold relative to an IgG), but the rest of them either had no fold when they were supposed to or the values for the IgG and the specific antibody were almost the same. This was weird because the IgG/specific antibody values were even higher than the values for the specific antibody in those other genes which resultes well...
-In the other PCR I couldn´t get any fold, in this one I included a positive control: DNA obtained with agarose beads which worked well on PCR at some time back... and so did now.
No, I don´t think I should heat up my chromatin, right?...
Thank you very much for your suggestions!
Ok problem solved!!. Thank you everyone for your feedback!!
Apparently keeping the last buffer on ice was the worst idea ever...
Quick washes and buffers at RT are the key points here.
Hi Ana, I just had exactly the same problem and had to abandon my ChIP experiment because of it! The protocol I have said to keep the buffer at 4oC and I ended up with this thick gel that you described. Did you store the LiCl buffer in the fridge and then get it down to room temperature before you used it? Could you possibly send me the protocol that worked for you in the end?
In my ChIP experiments I get up to chromatin from 25 million cells and I wash twice with COLD LiCl buffer (and also 0.5% NP-40) and I've never encountered any problems. My washes consist of 1ml buffer and 5min agitation in 4oC.
Hi Ana, actually I'm having the same problem with my dynabeads coagulated during the washes. So, do you believe that keeping the wash buffers at RT solves the bead-coagulation problem?
BTW; I wonder if you have Sarkosyl in your sonication/lysis buffer.
I believe pre-clearing chromatin and removal of insoluble fraction after sonication (centrifuge sonicated sample at maximum speed- 16 K )will help to overcome this problem. I usually keep my all washing solution in Cold room and do washes at cold.
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