Histone extraction HLB protocol

Sigrid Verhelst

Published: 2024-01-23 DOI: 10.17504/protocols.io.x54v9p49pg3e/v1

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only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind.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.

Steps

Isolate nuclei

Start from a dry (snap-frozen) cell pellet

Add hypotonic lysis buffer (HLB) to cell pellet: 200 µL for 1x106 cells ⁶cells 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

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 ⁶ 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

10.

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)

11.

Incubate on ice for 30min

12.

Spin for 10 min at 4°C and 16.000 g to pellet the histones

13.

Remove the supernatant

Note

Be careful: the pellet is not always visible

Wash steps

14.

Add ice-cold acetone (do not resuspend the pellet) to remove TCA, make sure the pellet is fully covered with acetone

15.

Spin for 5 min at 4°C and 16000 g

16.

Remove the supernatant

17.

Add cold acetone again (do not resuspend the pellet) to remove TCA

18.

Spin for 5 min at 4°C and 16000 g

19.

Remove the supernatant

20.

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)