Please help me with my sonication (gel included)... - I know, everyone asks the same thing, but I'm desperate (Apr/08/2008 )
I've been having trouble with my ChIP so I've gone back to the basics of trying to optimize my sonication. I am starting with Xenopus embryos so my initial steps are a bit different than what most of you are probably familiar with, but my sonicated DNA should be the same.
After fixation I homogenize my embryos with 10 strokes in a Dounce in buffer with 50mM Tris pH 8.0, 10mM EDTA, and 150mM NaCl, plus protease inhibitors from Upstate. I then immediately sonicate with a Fisher Dismembranator 500 for 6 pulses (2x15%, 2x17%, 2x19%) of 10s with 50s between pulses. Sonication is done in an ice bath. I then spin the sample at high speed in a microfuge for 20 minutes to remove lipid and debris, transfer the supe to a new tube, and add SDS up to 1% final. I then repeat the sonication exactly as above, but with only three pulses (15%, 17%, 19%).
My results are shown on the following gel. I took 5ul of the sample after adding SDS, and after each of the last three sonications. I then added 5ul of RNAse for an hour at 37, then 1ul of the Upstate Proteinase K for 2 hrs at 62, followed by 10 minutes at 95. I loaded 10ul of each sample.
My big question is obviously about the quality of my sonication, but more specifically, what is that 'front' running at about 250bp?
I am not familiar with your protocol but why don`t you add SDS in the first sonication?
I believe the idea is to break up the nuclei and free the chromatin with the first round, without solubilizing the lipids and yolk that are abundant in Xenopus embryos. The subsequent spin removes these and then the SDS is added to aid in shearing the chromatin.
If you agree that there is a problem with my DNA, I'm quite open to modifications. Perhaps I need to do the initial sonication to break up the nuclei for fewer pulses.
I was just asking
Were your shearing pictures looking like this before?
Not exactly, but I've been modifying things so much that it's hard for me to compare. I wasn't treating the samples with RNase previously, instead just incubating with proteinase K at 62 for 2 hours and purifying over a column as suggested in the Upstate MagnaChIP protocol.
I modified my chromatin cleanup to follow Dave's protocol here: http://www.protocol-online.org/forums/inde...showtopic=34892
Here's what he suggested and what I am doing:
- take 5ul of sonicated sample (5ul from a total of 400ul containing 5 million cells).
- add 5ul RNAse Cocktail (Ambion, Catalog # AM2286 or AM 2288 depending on quantity, 5ul cocktail = RNAse A - 2.5 units,
RNAse T1 - 100 units). Incubate 2 hours at 37 degrees. 2hrs may be overkill perhaps, but I like to be sure.
- add 5ul Proteinase K (not sure about concentration, but it is Proteinase K from Promega, made up as per instructions). Incubate 2
hours at 62 degrees and 10 mins at 95 degrees.
- Samples are then ready to run on a gel, so don't bother with the clean up unless you are doing a PCR.
The only difference is that I am using 1ul of the proteinase K from the Upstate kit rather than using the Promega Proteinase K that he has. I think the concentrations are about the same.
Uff, I know that!! It was the same with me in the beginning. But you have to start somewhere, right?
Here are my suggestions (although there are many protocols and they result differently in different hands. So, if you think I am telling some bullshit, just ignore it):
1. Have you seen a difference in the shearing picture with/without RNaseA? Is the smear reduced with RNAseA treatment?
2. I do the incubation with proteinaseK at 65 degree for at least 4h. Is 62 degree the given temperature for your active proteinaseK?
3. You use 5ul of your sample. Do you think that there is enough DNA? Maybe you could use a bit more of your sample (let's say 10ul). Compared to your ladder, the sample material looks faint -some cells give more DNA, some cells less.
4. Have you ever tried to run the gel for 30min (kind of short run)? The longer you run the gel, the longer the smear size will appear (though the ladder looks nice).
5. Are you sure that the crosslinking works properly (not overcrosslinking or not crosslinking)?
When I look at your gel picture, I see the smear between 250 and about 1000 bp. This size range (or better 500-1000bp) is recommended mostly in protocols. Are you expecting something else? Maybe you can attach a better gel picture if you have since I don't know how your gel should look like.
I think if you would add some more DNA and run the gel a bit shorter, your gel will look nicer (at least worth to give a try).
Coming to your problems with the ChIP results... Ok, you did not tell what kind of problems you have (no signal, weak signal, high background, strong signal in your negative control etc.) but... Are you sure that the antibody and the beads are still allright? I guess, the buffers and everything also have been checked. If you would give some more details, we might be more helpful??
That front you are seeing could be because you are getting down to the size of mononucleosomes. Your fragmentation looks good to me. If you're having problems with ChIP, it's not because your fragment sizes are off. What kind of problems are you having? What are you trying to IP?
I found my gel looked more attractive if I did a PCR clean up, it got rid of the fat bands I had in the middle of my smear, but still the smear looked the same in terms of size of DNA fragments.
When I took 5ul from my 400ul containing 5 million cells, there is approximately 62500 cells in that 5ul, so make sure you are loading roughly the equivalent amount of cells.
The only other thing I can think of is that perhaps your cells weren't lysed properly. You are however homogenising but perhaps Cell Lysis buffer works better? I incubate for 15 mins on ice in the Upstate Magna ChIP kits 'Cell Lysis buffer' before sonication. I have noticed when I was playing around that if you didn't do this then it certainly made a difference. I even saw a few gels that looked similiar to yours when this was the case. I had smears that went the entire length of the well but I don't believe this was actually sonicated DNA I was seeing.
Hope you can sort this out soon!
Well, I was previously getting pull-down with my Txn Factor antibodies, well over the IgG control, but the results were never quantitatively consistent. I was seeing %Input ranging from 0.1% to 2%. Qualitatively, they were reproducible (some antibodies IP's, some didn't, consistently). My IgG never pulls down so I can't quantitate as fold-over-IgG.
But now I am just not getting any IP at all, from any of my antibodies. I am concerned that my inconsistent results were due to being just on the borderline of detectability as my Ct values were usually in the mid- to high 30s and when diluted out even the input triplicates weren't very reproducible with Ct values in the high 30s.
I've tried cleaning things up by spinning the samples after the incubation with antibody and prior to adding the beads. I've switched from the MagnaChIP kit to the agarose bead kit in the hopes of getting more sensitivity, but my first attempt with the agarose beads left me with no IP either. So I decided to go back to first principles and work everything through carefully.
Another interesting observation is that I seemed to get the best results when my sonication foamed. This led me to wonder if I have been oversonicating when my samples don't foam, thus my current focus on the sonication. I was originally hand-holding my samples during sonication, but I rigged up a ring stand to hold the eppi tube in an ice bath, both to be more consistent between runs and to help keep the samples cooler. I decided to just throw away foamed samples since it seemed like an easily controllable variable, but my IPs haven't really worked since then, at least not with any robust pull-down.
Thanks for any ideas you might have.
If you look closely at my gel, it does seem that the smear runs the whole length of the gel. I thought this was normal and that I should focus on the brighter, mean size of the smear, but maybe that is leading me down the wrong path.