I've been performing ChIP-seq experiments using the Abcam antibody for H3K27 acetylation. I use a 1% SDS buffer to sonicate and then dilute the sample down for the IP. I have used different SDS concentrations in the IP but so far 0.4% seems to give the best results (large enough library concentration and strong enrichment), however I've read that most people dilute their samples down to
In your case there are two things to think about when selecting a buffer for IP. Ability of Ab to bind antigen and effect of buffer on ability of antigen to bind DNA. Both of these can be strongly affected by your lysis buffer and your IP buffer (usually the same). For example, when I am doing Co-IP, I will often use a mild detergent, such as NP-40, because I don't want to affect protein-protein interactions. Unfortunately, this is something that has to be determined emperically. That being said, the two protocols I have for ChIP only have SDS concentrations of 0.1%. There are numerous protocols out there though, I would take a look on-line and see what you can find.
Szak RIPA buffer 50 ml
150 mM NaCl 1.5 ml (5 M)
1% v/v Nonidet P-40 5.0 ml (10%) or 0.5 mL (100%)
0.5% w/v deoxycholate 2.5 ml (10%)
0.1% w/v SDS 0.25 ml (20%)
50 mM Tris pH 8 1.67 ml (1.5 M)
5 mM EDTA 0.5 ml (0.5 M)
Right before use:
Protease Inhibitors Cocktail
Phosphatase Inhibitors: 50 mM NaF/ 0.2 mM sodium orthovanadate
HDAC inhibitors: 5 mM trichostatin A/ 5 mM sodium butyrate
0.5 mM PMSF
Mammalian Cell lysis buffer
10 mM Tris-Cl pH 7.5
10 mM NaCl
3 mMMgCl2
0.5% (v/v) NP-40
Store up to 1 year at 4◦C
Hello.
Thats a great answer Michael. I have been doing IP and I think you can final SDS concentration to 0.1 or
The SDS is required for efficient shearing of the DNA in your prep. The trade off being indeed the efficiency of the IP.
For some antibodies, 0.5% SDS is fine for the IP and is great for removing excess background. Others do indeed require less than 0.1% SDS. For our ChIP experiments, after the sonication, we add Triton X100 to final concentration of 1%. This helps quench the SDS.
A good resource to follow for ChIP in general is at the following link. There are three parts which explain the different steps.
https://www.whatisepigenetics.com/collecting-analyzing-libraries-for-chromatin-immunoprecipitation/
In general, if your IP works and you get nice signal, the concentration of SDS is not important. It is very much an empirical process where each antibody needs to be optimised. Antibodies recognising modifications to Histones are very good - and I can recommend Active Motif H3K27Ac, H3K27me3, H3k4me3 and H3K4me1. These have worked really well. The abcam H3K27Ac also works well.
Good luck.
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