Protocols:
Extracts for Assays of NFAT Complexes from Cortical Neurons |
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.
Small Scale Nuclear
and Cytoplasmic Extracts for Cortical Neurons
1. Pre-cool the rotor, tubes and buffers in the cold room overnight. 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 Cell Lysis- Be certain to add the porteinase inhibitors and freshly made DTT just before starting! 1. Aspirate medium from plates at rt, 2. Gently and quickly rinse with room temp PBS and aspirate the PBS off quickly and gently. Place plates on ice and add 1 ml of Buffer A (ice cold) on the plates. At this point transfer everything to the cold room. 3. Scrape the cells off the plates with a cell scraper, use a fresh scraper for each stimulation condition and pipet the cells into an Eppendorf tube. Check the nuclei with trypan blue ( you should see oval , dark blue nuclei and no intact cells) 4. Spin in eppendorf at a setting of 3 for 3 minutes. 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. 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. 8. The solution should become thick and viscous as the nuclei break and the nuclear proteins are released. Transfer to a TLA-100.2 ultracentrifuge tube. Since the solution is viscous you have to use a wide orifice tip or cut off the tip from a blue tip to transfer into the ultracentrifuge tube. 9. Spin 10-15 minutes at 100,000 rpm at 4 degrees C. To continue cytoplamic prep. Skip to 11a. 10 Transfer 300 ul of the supernatant to a second TLA-100.2 tube. 11 Add equal volume of 3 M ammonium sulfate. Pipette to mix.. 11a. For cytoplasm, remove 800 ul supernatant and add 0.3 g/ml (NH4)2SO4 and rotate in the cold room until the Ammonium sulfate is completely dissolved 12. Spin 10 minutes at 100,000 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 of 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 for cytoplasmic and 30-50 ml for nuclear extract 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. |
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