Nuclear Extract and Cytoplasmic Fraction Preparation protocol for ADAMTS13 Antibody (NB100-584):
Nuclear Extract and Cytoplasmic Fraction Preparation:
1. Nuclear extracts (NE) and cytoplasmic fractions (S100) were prepared by Dignam's method (Dignam, Lebovitz, and Roeder, Nucleic Acids Res. 11: 1475-1489. 1983).
2. 100 liters of HeLa cell culture were harvested and washed 3 times with cold PBS.
3. The packed-cell volume (PCV) was measured, and the cell pellet was gently resuspended with 5 PCVs of hypotonic buffer (10 mM HEPES-KOH [pH 8], 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.2 mM PMSF).
4. Cells were incubated on ice for 10 minutes and then pelleted by centrifugation at 1,800xg for 10 minutes.
5. Hypotonic buffer was added to 2 PCVs, and cells were resuspended and then homogenized with 15 strokes using a pestle B in a Dounce glass homogenizer until the cells were more than 90% lysed, as determined by a light microscope.
6. The lysate was centrifuged at 20,000xg for 30 minutes at 4 degrees Celcius.
7. The supernatant was saved for S100 fraction, and the pellet was saved to measure the packed nuclear volume (PNV).
8. 0.4 ml of extraction buffer (20 mM HEPES-KOH [pH 8], 0.6 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% [vol/vol] glycerol, 1 mM DTT, 0.2 mM PMSF) per ml of PNV was added.
9. Cell nuclei were homogenized with 10 strokes of pestle A in the homogenizer.
10. Suspension was stirred at 4 degrees Celcius for 30 minutes and centrifuged for 30 minutes at 20,000xg.
11. The supernatant (nuclear extract) was aliquotted for use.
12. The S100 fraction (resulting supernatant) was mixed with 0.11 volume of high-salt buffer (20 mM HEPES-KOH [pH 8], 1.2 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20% [vol/vol] glycerol, 1 mM DTT, 0.2 mM PMSF) and centrifuged at 100,000xg for 60 minutes at 4 degrees Celcius.
13. This supernatant was dialyzed for 2 hours at 4 degrees Celcius.
14. The sample was centrifuged for 30 minutes at 20,000xg and the supernatant (S100) was aliquotted for use.
Immunoprecipitation Antibody Characterization:
1. HeLa NE and S100 were diluted with 1 volume of RIPA buffer [150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris [pH 8]).
2. Cleared by spinning at 100,000 g for 20 minutes at 4 degrees Celcius.
3. 1 ml of supernatant (~10 mg total protein) was mixed with 20 ug of primary antibody (NB 100-584) and rotated overnight at 4 degrees Celcius.
4. Supernatant was mixed with 0.05 ml of protein A-sepharose beads (50% slurry) and rotated for 2 hours at 4 degrees Celcius.
5. Immunoprecipitates were washed 3 times with the 10% RIPA in PBS.
6. The washed beads were boiled with 0.04 ml of Laemmli buffer and subjected to SDS-PAGE (4-20% Tris-glycine gel).
Complex purification:
1. NE and S100 were cleared by spinning at 20,000 g for 30 minutes at 4 degrees Celcius.
2. 1.5 ml of supernatant (~15 mg total protein) was mixed with 20 ug of primary antibody (NB 100-584) and rotated for 4 hours at 4 degrees Celcius.
3. Sample and antibody mixture were centrifuged at 15,000 g for 20 minutes at 4 degrees Celcius.
4. Supernatant was mixed with 0.05 ml of protein A-sepharose beads (50% slurry) and rotated for 1 hour at 4 degrees Celcius.
5. Immunoprecipitates were washed 3 times with the NETN buffer (20 mM Tris-HCl [pH 8], 100 mM NaCl, 1 mM EDTA, 0.5% NP-40).
6. The washed beads were boiled with 0.04 ml of Laemmli buffer and subjected to SDS-PAGE (4-20% Tris-glycine gel).
*If an insufficient amount of protein is purified for identification from 15 mg of extract, carry out the same procedure using 50-100 mg of extract to increase the amount of purified protein yield.
