ChIP - no immunoprecipitated DNA - (Dec/25/2005 )
Hi,everyone!I need your help!
I did chip for 3 times with upstate kit ,but i couldn't immunoprcipitate any dna/protein.I am very puzzling,please help me .the main procedure is following:
First,i'm sure that the sonication is no problem.The sonicated-chromatin was about 300-400bp,and i proved it in an agarose gel.Second,i added 10ul antibody(it can use for chip,from upstate )for immunoprecipitation and incubated overnight at 4oC.Third,in the washing step, i rotaated the agarose/antibody/histone complex in ice for 3-5minutes after adding washing buffer each and pelleted it at 1000rpm at 4oC ,30seconds. Furthermore,i could see the white pellets although it's small in ethanol precipitation . i used the reagent helping nucleic acid precipitate in this step.
But ,i couldn't see anything in western blot.Of course,pcr was no products.i also measured the OD260 value of immunoprecipitated-DNA,but to my surprise it's minus.
I don't know in which step DNA is lost,or where i made a mistake.help me!
may I know your PCR cycle numbers? Maybe it was not enough.
But I guess you need to solve the western problem first, you said you found nothing there, how about input control?
hi bullfrog,glad to see your reply.
I usually did 30 PCR cycle numbers.Do you know how many cycle numbers is the best?
I did have probelm in western,because I couldn't find anything even if I used protein marker as sample.I suspect DNA didn't crosslink with protein .Do you think it is possible?
sounds like your antibody to the protein of interest is not binding to your samples, or that your samples do not have detectable levels of the protein you are trying to look at.
This has happened to us in the past, you should check that your cells/tissues have the transcript for your protein and that it is present in your whole cell lysate on a western.
I also think that cell numbers was not enough.The protocol says that 1X106numbers is ok,but i used to 1X107.Apparently,it was too little.Do you think how many cells is right?Moreover,my protein of interest is histoneH3.I am studying H3-lys methylation.
You said that cells must have the transcript for my protein.I don't konw that.Can you tell me how to do it?Thank you.
10^6 cells is plenty as a starting point.
if you are looking at Histone H3 there shouldn't be a problem in terms of amounts unless it is one of the new histone H3 variants. If you are looking at lysine mehtylation I would assume you are either looking at lysine 9 or lysine 4, or lysine 27 methylation of histone H3, if so it should be quite easy to IP.
I would say that your antibody is not recognising the epitope you are after, where did you source the antibody from? Another problem could be overcrosslinking your chromatin for IP, try and reduce your fixation times, because histones are tightly packaged i have read that it is not necessary to cross link however I have not tried it hands on.
My antibody was bought from upstate.It belongs to chip grade.So it should be no problem.You said it was possible to overcrosslink the chromatin for ip,but,how could overcrosslinking affect chip?Is it necessary to restrain crosslink?
In addition,can you tell me which grade formaldehyde you used in chip is?I always used common chemic grade,but my boss said that i should use EM grade.He thought that EM grade was the best for chip.Can you agree?
Last time,you said that I should do the transcript for my protein.I want to know how to do transcript.Please tell me.Thanks.
Happy new year!
If your antibody is ChIP grade then it should work, however double check the batch number with Upstate and see if other people are having problems with that antibody of that particular batch (if it ain't working for western, something is up)
when you say your western was not working, you did not detct any bands at all? or were there bands of unexpected size? you may need to acid treat your histones to dissociate the nucleosome before running a PAGE gel.
You can overcrosslink your chromatin and this can mask your epitope beyond recognition, less crosslinking time is better. As for formaldehyde, if you can afford it, use EM grade, we have used this in the past with no problems and it is great as it comes prealiquoted for single use. However the normal stuff from sigma is fine, however you should be aware that it is in methanol/water solution to keep it stable as methanol is also a fixative as well this would affect the crosslinking (for in less time). I don't think EM grade contains methanol check out this website here. so in that respect your PI is correct to say EM grade is better.
As for transcript levels, you would only be measuring Histone H3 transcription, unless it is a variant like CENP-A or H3.3 say, would be measured by quantitative RT-PCR.
Someone told me that the beads should be pre-absorbed,but my beads was bought from upstate.I bought the chip kit(Catalog #17-295) from upstate.It contains Salmon Sperm DNA/Protein A Agarose, Catalog #16-157C, Lot # 27977. Its discripition is following:
One vial containing 1.5ml packed beads with 600μg sonicated salmon sperm DNA, 1.5mg BSA and approximately 4.5mg recombinant Protein A. Provided as a 50% gel slurry for a final volume of 3ml per vial. Suspended in TE buffer, pH 8.0, containing 0.05% sodium azide. Liquid suspension.
I thought that the beads had been pretreated,but they suggested me pretreating the beads again.So,I decided to pretreat its ,however,I didn't find out detailed pretreatment protocol.Hope that you can give me good advice.Thanks.
wow that is pretty cool how you can buy your beads pre-treated.
we normally pre-anneal our beads in RIPA buffer (the buffer used to lyse cells to release chromatin) and salmon sperm DNA so that the buffer in the beads was equivalent to the chromatin soup.
there is a few detailed protocols on the abcam website here is the link for X-ChIP