The document outlines a protocol for preparing nuclear extracts on a small scale, detailing steps from cell spinning to dialysis and supernatant collection. It includes specific buffer compositions and conditions for various stages of the preparation, as well as instructions for handling nucleosomes and histone proteins. Additionally, it provides a recipe for assay buffers necessary for histone methyltransferase and demethylase activities.

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.