Your ChIP input material is cross-linked, and thus bands are not likely to run at their expected MW (ideally much of your factor is cross-linked to DNA and/or other proteins). It would be far more instructive to check whether you can detect your protein by Western in a nuclear fraction of non-crosslinked material. In the event you can succeed there, I would proceed with the ChIP experiment using the sonicated material you prepared and not worry about trying to use ChIP input material for Westerns. Since your positive control looks OK, I don't see any reason to question the transfer step.

Thanks a lot Daniel.

Ok this was extremely stupid of me that i didn't think about the "crosslinking" problem...

it is pretty obvious that since my sample is Crosslinked, i am not going to get bands at the expected range.

But i was thinking that if the problem is "crosslinking", then what if after i take the input, "reverse crosslink" it without using "proteinase K" and then then run the sample in western.

How about that?

that will theoretically rule out the problem.

so will it be better to check input protein by the following process:

1. fix cells by formaldehyde and glycine

2. sonicate the sample @ 20cycles

3. make up the volume upto 2ml with ChIP dilution buffer

4. pre clear with beads and take 100 ul from that sample as input.

5. overnight reverse crosslinking with NaCl but No PRO K.

6. run that sample as INPUT Protein sample.

Thanks a lot Lilien.

But just wanted to say that my beads are not antibody conjugated. i first incubate the sonicated sample with Antibody to induce Protein-Antibody binding. then i put the beads to induce Antibody-Protein binding.

the very fact that i am also passing the sonicated sample through the same beads is because i am using Agarose beads. thus it is reported to have nonspecificity. the Pre clearing will remove anything that has an inherent affinity for the beads. so that after i add the beads again for antibody binding, it does not create non specific pull down.

We've tried this and unfortunately reversal of crosslinking is highly inefficient. It is adequate in combination with Proteinase K to liberate enough DNA to permit the ChIP assay to work. However, even after so-called reversal of crosslinks overnight at 65C, your proteins still will not run at the correct MW on SDS-PAGE. So I cannot recommend this. The best you can do is dot-blot, but you lose any information as to whether your Ab is recognizing the expected antigen based on molecular weight. If you have a negative control (e.g. knockout), you might be able to interpret it.

I think you need to troubleshoot several aspects of your issue and who didn't have.

Normally, if you boil your crosslinked sample in SDS buffer with DTT or 2-ME for 10 or 15 min it should be OK.There are papers on that and in our lab it works.

I suggest you do the following

1/ prepare nuclei but keep the cytoplasmic fraction

2/ after sonication keep the pellet

3/ proceed with ChIP but disolve you ChIPped sample in SDS buffer with DTT and boil

Into the gel load samples from cytoplasmic, sonicated DNA (input), your ChiPped chromatin and also the pellet. Your protein is somewhere

It's possible that your protein doesn't crosslink to the DNA (some transcription factors don't crosslink to the DNA with formaldehyde you need to double crosslink, other chromatin associated factor also)

Another issue is that your protein is associated with chromatin regions that are hard to release. Analyzing the pellet would tell you

Good luck!

Thanks a lot Abdelhalim.

But just wanted to ask whether not adding DTT to the fixed and sonicated sample affect protein detection in western?

the way i am preparing my input sample for western is as follows.

1. add 5x loading dye to appropriate volume to make it 1x

2. heat the input sample for 5 min @ 100*C

3. invert mix and quick spin

4. load and run for western

I have never added DTT for whole cell lysate protein preperation. that is why i haven't been using it for ChIP input samples.

will that make a difference? :/

I think yes! for denaturing PAGE you must add DTT or 2-Me to prevent the oxidation of free thiols groups (cystein and methionine). I guess you're not using it to avoid the interference with denatured antibody heavy chains (~ 55kD) but there are ways to get around that!

Anyway I am not aware if it has to do with the chemistry of reverse crosslinking.To my knowledge the rate of decrosslinking is a function of temperature only. You have to detect it in someway

Hi,

Thank you so much for all these information.

I have the same problem with the ChIP protocol, before cross-linking the transcription factor can be detected in westernblot but not after cross-linking. So I assume the ChIP won't work because the antibody we used for western blot and IP are the same one, if the antibody didn't properly recognize the cross-linked transcription factor then the IP beads with same antibody should not work either, right?

Also Aritra, may I ask if you have any ideas now about how to solve this problem?

Didi

The only way to solve these problems is ChIP-PCR. If you have a positive control then you are good to go, but if you dont, then generate very high quality primers around sites that have consensus binding sequences.

Best of luck

@Aritra Misra hi Aritra, I need to do the same thing. Did this work for you?