Extracts for Assays of NFAT Transcription Complexes
Gerald R. Crabtree Sept 1985
Modified by Katie Ulman and Mike Flanagan 1991
Updated Sept 2001

NFAT transcription complexes can not be detected in a conventional Dignam nuclear extract. The following method was developed to allow maximium extraction of NFAT complexes and is taken from Durand et al. MCB 1988 and Shaw et al. Science 1988.
Solutions and Materials
Buffer A
25mM Hepes ph 7.0
25 mM KCl
0.1 mM EDTA
5mM MgCl2 (for some cells 10- 12.5 mM are necessary to protect the nuclei
10% glycerol
1mM DTT
+ protease inhibitors (antipain, aprotinin. benzamidine, leupeptin, pepstatin, PMSF), phosphatase inhibitors (b glycerol Phosphate, PNPP)
Buffer B
0.3 M Hepes ph 7.6
1.4 M KCl
30 mMMgCl2
DTT 1mM
PMSF mM
Buffer C
50mM Hepes ph 7.6
50 mM KCl
0.1 mM EDTA
1 mM DTT
PMSF 1 mM
Glycerol 10 %
Ammonium Sulfate (NH4)2SO4 3 M (pH 7.9)
PBS
Centrifuge Tubes This protocol was written for the 2 ml tubes for Beckman mini-ultra centrifuge TLA 100.2 tube, or polycarbonate 7/16 inch by 1 3/8 inch tubes (343770)

Procedure
1. Pre-cool the rotor, tubes and buffers in the cold room overnight.
2. Wash 3-5x107 cells in PBs and resuspend in 1 ml Buffer A.
3. Spin in eppendorf at a setting of 3 for 3 minutes.
4. Resuspend in buffer A plus 0.05% NP40 (NP-40 concentration may have to be varied for different cell types. Check under the micrscope to see that you get complete lysis).
5. Incubate on ice one minute. Pipette up and down to suspend cells. Centifuge as before. Steps 4 and 5 can be repeated to clean up the nuclei.
5a For cytoplasm save supernatants. Add 1/10th volume buffer B. Skip to step 9.
6. Resuspend pelleted nuclei in 250 ul of buffer C; measure total volume with the eppendorf and add additional buffer C to a total volume of 315 ul.
7. Transferr to a TLA-100.2 ultracentrifuge tube. Add 35 ul 3 M ammonium sulfate to bring the final concentration to 0.3 M and rock for 30 minutes in the cold room. To reduce the volume one can use solid ammonium sulfate.
8. The solution should become thick and viscous as the nuclei break and the nuclear proteins are released.
9. Spin 10-15 minutes at 100,000 rpm at 4 degrees C. To continue cytoplamic prep. Skip to 11a.
10 Transfer 2 x 140 ul of the supernatant to a second TLA-100.2 tube.
11 Add equal volume of 3 M ammonium sulfate. Pipette to mix. Alternatively, to reduce the volume, solid ammonium sulfate can be used.
11a For cytoplasm, remove 800 ul supernatant and add 0.3 g/ml (HN4)2SO4
12. Spin 5-10 minutes at 50,000 rpm and 4 degrees C.
13 Remove the supernatant. Note: if you have scaled down this procedure and have a very small amount of protein, clear the wall fo the tube with either a flame-polished Pasteur pipet or with a cotton plugged yellow pipet tip instead of running a spin column.
14. Resuspend in 50-100 ul of Buffer C. Use a spin column (P6DG, Biorad) to desalt. Spin 250 g for 5 minutes. If you have access to a microdialysis apparatus it will give better results and more concentrated extracts.
15. Using the gel shift assay, check a constitutive DNA binding protein to be certain that it gives a sharp band and that the extracts are not degraded.
16. Freeze samples in liquid nitrogen or the –70 degreeC freezer.
Note: Preparation of Extracts from adherent cells, specifically cultured embryonic cortical neurons from mice.
Buffer A must be modificed to all full detachment of the cells from the plates. To allow more effective recovery of the cells, add EDTA to 0.1 mM and MgCl2 to 12.5 mM to Buffer A and physically remove cells with a scrapper.