Jan 23, 2024
Histone extraction HLB protocol
- 1Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium
Protocol Citation: Sigrid Verhelst 2024. Histone extraction HLB protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9p49pg3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 22, 2024
Last Modified: January 23, 2024
Protocol Integer ID: 93870
Funders Acknowledgement:
BOF
Grant ID: Mandate 01D23313
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Protocol to extract histone proteins using a hypotonic lysis buffer for isolation of nuclei prior to acid extraction of histones that is easily amenable to label-free MS owing to the lack of detergents in the lysis buffer.
Isolate nuclei
Isolate nuclei
Start from a dry (snap-frozen) cell pellet
Add hypotonic lysis buffer (HLB) to cell pellet: 200 µL for 1x106 cells and resuspend softly
Note
HLB buffer: 10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl2 supplemented with 1 mM DTT, 1 mM PMSF, Halt Protease and Phosphatase Inhibitor Cocktail 100x (78440) and phosphatase inhibitor cocktails II and III (P5726 and P0044, Sigma-Aldrich, 1 mL of cocktail for 100 mL of buffer)
Rotate for 30 min at 4°C to promote lysis of cell membrane (mechanical shear)
Pellet the nuclei by centrifugation at 10.000g for 10 min at 4°C
Discard the supernatant
Extract histones
Extract histones
Resuspend the pellet in 0.4N hydochloric acid (HCl) by soft pipetting until no clumps left in solution by adding 125 µL HCl for 1x106 cells (if necessary: vortex)
Incubate for 30 min in acid on rotator at 4°C to promote lysis of nuclei and solubilization of histones
Spin down for 10 min at 4°C and 16.000 g
Transfer supernatant to new Eppendorf (histones are present in the acid since they are alkaline proteins)
Isolate histones
Isolate histones
Add, drop by drop, trichloroacetic acid (TCA) until a final concentration of 33% is reached to promote precipitation of histones and invert the tube several times (results in a milky solution)
Incubate on ice for 30min
Spin for 10 min at 4°C and 16.000 g to pellet the histones
Remove the supernatant
Note
Be careful: the pellet is not always visible
Wash steps
Wash steps
Add ice-cold acetone (do not resuspend the pellet) to remove TCA, make sure the pellet is fully covered with acetone
Spin for 5 min at 4°C and 16000 g
Remove the supernatant
Add cold acetone again (do not resuspend the pellet) to remove TCA
Spin for 5 min at 4°C and 16000 g
Remove the supernatant
Dry at room temperature for 30 min (until no acetone left)
Note
Samples can be stored at -80°C or -20°C until further use (propionylation/digestion) or part of the sample can be prepared to perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Preparation for SDS-PAGE
Preparation for SDS-PAGE
Resuspend in MilliQ water (50 µl for 1x106 cells)
Transfer 400.000 cells to a new Eppendorf tube for gel-electrophoresis (optionally)
Note
If there are still clumps left: Spin for 10 min at 4°C and 16000 g and transfer the supernatant in a fresh Eppendorf
Vacuum dry the samples (SpeedVac)
Note
Store both Eppendorf tubes (for propionylation/digestion and SDS-PAGE) at -20°C or -80°C until further use