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.

Buffers for Nuclear ExtractBuffer A Stock 1L10mM 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 E50mM Tris 7.9 1M 40ml .25% Glycerol 100% 200ml0.5mM EDTA 0.5M 0.8ml .5mM MgCl2 1M 4ml5mM 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.