Jan 31, 2024
Direct acid extraction
- 1ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium
Protocol Citation: Sigrid Verhelst, Laura Corveleyn 2024. Direct acid extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x6odlqe/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 31, 2024
Last Modified: January 31, 2024
Protocol Integer ID: 94448
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only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind.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 histones from cells using direct acid extraction with the purpose of label-free LC-MS/MS measurement.
Materials
Protein lobind Eppendorf tubes
Thermoshaker
Cooled centrifuge (4°C)
Ice
MilliQ water
12 M HCL (dilute to 0.4N HCl: for 300 mL: 10 mL 12M (37%) stock + 290 mL milliQ water)
TCA powder (make 100% TCA solution)
Ice-cold acetone
Resuspend the pellet in 0,4 N HCl by soft pipetting until no clumps are left in solution (125 µL for 1x106cells)
Note
If necessary, vortex softly
Incubate 4h in acid on rotator 4°C to promote lysis of nuclei and solubilization of histones.
Spin for 10 minutes at 4°C, 16000 g.
Transfer supernatant to new Eppendorfs.
Note
Histones are present in the acid since they are alkaline proteins
Add, drop by drop (very slowly and in the middle of the eppendorf), TCA (final concentration 33%: 61.25µl for 1x106) to promote precipitation of histones and invert the tube several times.
Incubate on ice for 30 minutes.
Spin for 10 min at 4°C, 16000g to pellet the histones.
Note
Important: Place all the eppendorfs in the same position => makes it easier to know where the pellet is localized
Remove the supernatant.
Note
Be careful, the pellet is not always visible, smear on wall.
Add ±150 µL ice-cold acetone (don’t resuspend the pellet) to remove TCA.
Spin for 5 min at 4°C, 16000g.
Remove the supernatant.
Add ±150 µL cold acetone (don’t resuspend the pellet) to remove TCA. If not all the acid is removed it will destroy the SDS-gel.
Spin for 5 min at 4°C, 16000g.
Remove the supernatant.
Dry at room temperature for 30 minutes (until no acetone left).
Resuspend in milliQ water (50 µL for 1x106 cells).
Transfer 20 µL (from 400.000 cells) to a new Eppendorf tube for gel.
Vacuum dry samples.
Protocol references
Govaert E, Van Steendam K, Scheerlinck E, Vossaert L, Meert P, Stella M, Willems S, De Clerck L, Dhaenens M, Deforce D. Extracting histones for the specific purpose of label-free MS. Proteomics. 2016 Dec;16(23):2937-2944. doi: 10.1002/pmic.201600341. PMID: 27718312; PMCID: PMC5157773.