3 replies to this topic

[oldpostdoc]

Hi folks,

Does anyone have a ChIP kit they can recommend?
I'm just trying to do a standard ChIP using a chip-grade TF antibody on chromatin from adherent cells and analysing by qPCR. I have my sonication conditions worked out so don't want to use a MN-ase based kit. From browsing the forum it sounds like magnetic beads give lower background than, say, agarose. Is this a common view?

I'm new to ChIP and have been struggling along for a few months using my own buffers and an adaptation of an Abcam protocol given to me by a colleague, without success, so I feel it's time to give a kit a go to remove some variables. Any input gratefully received.

[Trof]

I'm using Sigma-Aldrich Imprint kit now (you can use sonication, MNase or both, it's not included in kit) but I can't say I had a great success with it.
My first results had indetectable some of the inputs (yes, really) even though immunoprecipitated DNA of the same samples were detectable. I strongly suspect the isolation columns to be the reason for this, they seem to be poorly made and drained too much of the elution buffer (and the elution volumes even differed between samples).
However the procedure, reagents and beads seem to be otherwise fine, since my coleague succesfully used the kit in past with different isolation columns. I'm going to try again soon with Qiagen columns for isolation.

[oldpostdoc]

Thanks for the feedback Trof.
The lack of responses to this post leads me to suspect that most 'practitioners' use their own reagents.

[Trof]

Well I repeated it now, and got very similar results with the inputs, so the main problem is somewhere else.

Otherwise I got used to the kit now, it takes about 9 hours to complete (in one day) and there dont seem to be any serious difficulties with it, apart from the columns. Anyway I found funny, that smaller kit contains 24 wells for immunoprecipitation but only 25 isolation columns.  Do they expect you to have 24 antibodies and just one input?
I had 4 samples and 4 antibodies, so I should be able to make 3 experiments if I have two kits, but that wouldn't be possible due to the lack of additional columns for inputs. Seriously, they have crapy columns and don't even put enough of them in there.