In mammalian cell fractionation experiments, it is often observed in protocols that after removing cytoplasm and nucleoplasm proteins, you digest your chromatin with either basemuncher or MNase to fragment the chromatin, then you spin, you got the supernatant which contains some loosely bound chromatin proteins and the pellet which contains lots of the tightly bound chromatin proteins. You would need to release those tightly bound protein by resuspend the pellet with high salt buffer. My question is since the chromatin has already been digested, why not all chromatin is in the supernatant after basemuncher digestion and spin? In other words, why the tightly bound chromatin proteins are not released into supernatant in this step?
Hi Han,
MNase only digests DNA at the euchromatin region and will not interfere with histones-DNA interactions. High salt is required to loosen the bond between DNA and protein.
Hope this helps.
Cheers
Pranay
Pranay A. M.
Hi Pranay, thanks for your answer. In that sense, if high salt is enough to loosen the bond between DNA and protein, then it is not necessary to fragment the chromatin because high salt itself will release chromatin bound proteins into the supernatant, is this right?
Thank you again.
Can you elaborate on your cell fractionation experiment's objectives and what you are interested in getting at the end?
Pranay A. M.
Thank you Pranay, I want to get Chromatin bound proteins, but I also want to know the principle in the protocol.
Following is the protocol which we use in the lab, everyone gets the chromatin fraction without problem, but no one really knows what's happening. The main question is still why it needs high salt (rather than low salt) to release the tightly bound chromatin proteins into supernatant? if it is the case as you said that high salt buffer itself could release chromatin bound proteins, then it is not necessary to use basemuncher to digest chromatin. I am confused.
Protocol:
Carry out all steps on ice and spins at 4oC.
- Resuspend cell pellet in 4x vol. SE buffer.
- Incubate 15mins on ice.
- Spin down nuclei 20mins @ max speed
- Take supernatant as unbound fraction.
- Wash nuclei by resuspending in 4x vol. SE buffer. Spin down again, 10mins @ max speed.
- Resuspend nuclear pellet in 2x vol CD buffer.
- Incubate 1hr on rotating wheel in cold room to digest chromatin.
- Spin down nuclei 20mins max speed
- Take supernatant as chromatin fraction 1. (If doing chromatin IP, use this fraction! Adjust resuspension volume depending on what you need).
- Resuspend pellet in 0.6x vol HS buffer. **Make sure to resuspend properly.
- Incubate 20mins on ice, agitate after 10 mins to prevent sedimentation.
- Add 1.4x vol. dilution buffer to bring salt back to 150mM
- Spin down 20mins @ max speed.
- Take supernatant as chromatin 2. Pool with chromatin 1 to get chromatin fraction.
- The remaining pellet will consist of “empty” nuclei, where most of the nucleoplasm and chromatin has been removed, apart from the stuff that remained insoluble after nuclease digestion and 500mM salt wash. Throw it away, or dissolve pellet in 8M urea and run on gel if you’re interested.
SE (soluble extraction) buffer
Extraction of cytosol and nucleoplasm
- 20mM HEPES-KOH, pH=7.5
- 150mM KAc
- 1.5mM MgCl2
- 0.5% NP40
- 10% Glycerol
- Protease and phosphatase inhibitors fresh
CD (chromatin digestion) buffer
- 20mM HEPES-KOH, pH=7.5
- 150mM NaCl
- 1.5mM MgCl2
- 0.05% NP40
- 10% Glycerol
- 250 U/ml basemuncher or bensonase
- Protease and phosphatase inhibitors fresh
HS (high salt) buffer
- 20mM HEPES-KOH, pH=7.5
- 500mM NaCl
- 1.5mM MgCl2
- 0.05% NP40
- 10% glycerol
- Protease and phosphatase inhibitors fresh
Dilution buffer
- 20mM HEPES-KOH, pH=7.5
- 1.5mM MgCl2
- 0.05% NP40
- 10% glycerol
- Protease and phosphatase inhibitors fresh
Buffers prepared and filtered, stored at 4oC.
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