Protocol for preparing small volume samples from ES cells, clinical samples and non-adherent cell types. This sample is designed to prepare samples for histone modifying enyme assays including demethylase, methyltransferase, deacetylase.

1. Nuclear Extract for small scale prep
 Spin cells 3K 10 minutes
 Measure packed cell volume
 Add 2 volumes buffer A
 Resuspend, Incubate 5-10 mins at
4oC
 Spin 3k 10 minutes
 Remove supernatant with pipet-this
is S100
Supernatant (S100)
 Add 10X buffer B to 1X
 Mix well
 Spin 30K 60 min
 Dialyse 1-2 hours against 20
volumes JHDM
 Spin 12K 20 min
 Keep supernatant
 Add Protease inhibitor tablets
(The nucleus) Pellet
 Add 1 volume buffer C, resuspend
 Rotate in cold 30 min
 Spin 30 min 12K Split into Nuc.Sup
and Pellet.
Nuc. Supernatant (soluble nuclear
proteins including transcription factors,
some histones)
 Dialyse 1-2 hours against 50
volumes JHDM
 Spin 20 min 12K
 Add Protease inhibitor tablets
Nuc.Pellet (nucleosomes, some
chromatin proteins)
 Resuspend with 1 vol buffer E
 Sonicate for 12 mins (30sec
on/30sec off)
 Spin 20 min 12K
 Keep supernatant, discard pellet
OR when active protein is not needed
Resuspend Pellet in 1M Guandium Chloride
(or 1M UREA). Start at 1 volume and
increase gradually until pellet is solubilized
(all solids gone) the solution will be cloudy.
Spin 12K for 30minutes.
Solution should be clear if not re-spin for
30mins.

2. Buffers for Nuclear Extract
Buffer A Stock 1L
10mM Tris 7.9 1M 10ml .
1.5mM MgCl2 1M 1.5ml .
10mM KCl 2.5M 4ml .
0.5mM DTT 1M 500ul .
0.2mM PMSF 100mM 2ml .
Buffer B (10X)
0.3M Tris 7.9 1M 240ml .
1.4M KCl 2.5M 448ml .
30mM MgCl2 1M 24ml .
Buffer C
20mM Tris 7.9 1M 16ml .
25% Glycerol 100% 200ml .
0.42M NaCl 5M 67.2ml .
1.5mM MgCl2 1M 1.2ml .
0.2mM EDTA 0.5M 320ul .
0.5mM PMSF 100mM 2ml .
0.5mM DTT 1M 500ul .
Buffer E
50mM Tris 7.9 1M 40ml .
25% Glycerol 100% 200ml
0.5mM EDTA 0.5M 0.8ml .
5mM MgCl2 1M 4ml
5mM DTT 1M 500ul .
0.2mM PMSF 100mM 2ml
BC buffers
[Final] Stock 100ml 500ml 1L 4L
20mM 1M Tris 7.6 2ml 10ml 20ml 80ml
0.2mM 500mM EDTA 40ul 200ul 400ul 1.6ml
10mM b-mercapto 70ul 350ul 700ul 2.8ml
10% Glycerol 10ml 50ml 100ml 400ml
[KCl]variable 2.5M KCl 0 0 0 0 BC0
4ml 20ml 40ml 160ml bc100
12ml 60ml 120ml 480ml bc300
20ml 100ml 200ml 800ml bc500
JHDM assay buffer: 50 mM HEPES [pH 8.0], 100 mM [NH4]2[SO4]2, 5% glycerol, and
0.2 mM PMSF ADD 1 mM a-ketoglutarate, 2 mM ascorbate when ready to do assays
For histone methyltransferase activity Add 30uM S-Adenosyl-L-Methionine to same
buffer. Alternatively use a HMtase buffer (100 mM Tris, pH 7.6/0.1 mM EDTA/10 mM
MgCl2/100 mM NH4Cl/1 mM DTT). NOTE the histone demethylases will not function in
this buffer.