Direct acid extraction
Sigrid Verhelst, Laura Corveleyn
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Abstract
Protocol to extract histones from cells using direct acid extraction with the purpose of label-free LC-MS/MS measurement.
Steps
Resuspend the pellet in 0,4 N HCl by soft pipetting until no clumps are left in solution ( 125 µL for 1x106cells 6cells)
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.
Add, drop by drop (very slowly and in the middle of the eppendorf), TCA ( final concentration 33%: 61.25µl for 1x106) 6) 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.
Remove the supernatant.
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 6 cells).
Transfer 20 µL (from 400.000 cells) to a new Eppendorf tube for gel.
Vacuum dry samples.
