Hi everyone. I need to read about protocols for preparing tissues before using them to measure epigenetic histone marks. I've been told these may be based on the 'Marburg protocol', however, I have not been able to find much info about this. Would really appreciate any help.
Dear Kevin Llinás-Caballero,
In my opinion, it is hard to extract all the histones when you using RIPA or another common lysis buffer to get the whole cell lysates, it is likely dependent on the chromatin status. For example, when you treat the cells with DNA damage reagents, the chromatin becomes loose and you may get more total histones using RIPA buffer.
So I recommend you to use an acid extraction method to get the histones.
Good luck
HISTONE EXTRACTION PROTOCOL
1. Harvest cells and wash twice with ice-cold PBS. PBS can be supplemented with 5mM Sodium Butyrate to
retain levels of histone acetylation.
2. Resuspend cells in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100 (v/v), 2mM
phenylmethylsulfonyl fluoride (PMSF), 0.02% (w/v) NaN3) at a cell density of 107 cells per ml.
3. Lyse cells on ice for 10 minutes with gentle stirring.
4. Centrifuge at 2000rpm for 10 minutes at 4°C. Remove and discard the supernatant.
5. Wash the cells in half the volume of TEB and centrifuge at before.
6. Resuspend the pellet in 0.2N HCl at a cell density of 4x107cells per ml.
7. Acid extract the histones overnight at 4°C.
8. Centrifuge samples at 2000rpm for 10 minutes at 4°C.
9. Removed the supernatant and determine protein content using the Bradford assay.
10. Store aliquots at -20°C.
Article Extraction, purification and analysis of histones
At the end of the experiment, euthanize mice by CO2. Excise tumor and organ samples and immediately snap-frozen in liquid nitrogen and preserved at −80°C until further use. Make tissue lysates using 2x lysis buffer (1 M Tris-HCl pH 6.8, 10% SDS, glycerol, β-mercaptoethanol, bromophenol blue). In brief, add 5 mm3 piece of tissue to 500 µl of 2x lysis buffer, homogenized for 30 seconds (PRO200, Biogen, USA), followed by 30 seconds sonication, heat at 95 c, 5 min, and centrifuge at 12,000 rpm for 5 min at 4°C. Supernatant was further diluted 5x, sonicated for 30 seconds, and heated at 95°C for 5 min. Centrifuge the sample at 13000 rpm for 5 min and use it as whole tissue protein lysate.
The protocol is perfect and works for all tissue types. Balkrishna Chaturvedi
2x lysis buffer also can be used to lyse cells cultured in vitro. Directly add 200 µl, 2x lysis buffer to plate (one well in 6 well plate, about 80 % confluent) and harvest using cell scraper, sonicate for 30 sec, and heat at 95 c for 5 min. Add 10 µl sample in one well to run the gel.
Limitation: The protein estimation
You need to adjust the buffer volume according to the cell confluency. After the first gel run, adjust loading by ponceau staining of the membrane.
2x Loading sample buffer (50ml)
1 M Tris-HCl pH 6.8 5 ml
10% SDS 20 ml
Glycerol 10 ml
β-mercaptoethanol 2.5 ml
Bromophonol blue 1mg
dH2O to 50 ml
Protocols will be different for different tissue types. As a rule, you can get high quality ChIP-seq data only for samples consisting mostly of the same cell type (say liver). If your tissue consists of comparable quantities of several cell types ChiP-seq readings from all of them will overlap and produce noise-like signal. So you need to find a way to separate different cell types first. For liver and tumor tissue you can find good protocols in David Waxman papers (Boston University).
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