- Proteins and Peptides
- Lysates and Cell Lines
Protocol for Immunoprecipitation Reactions:
Buffer preparation: make fresh on the day of immunoprecipitations
A) 5X PIC: Need ____ ml for IP Wash and 60 ul x ____ samples = _____ total
0.5 M EDTA (pH 8.0, RT) 50 ul X ____ml = _____
1 M Na2MO4 (4 degrees C) 25 ul X ____ml = _____
1, 10-phenanthroline (4.4mg/550uL ETOH) 18.7 ul X ____ml = _____
1 M NaF (4 degrees C) 200 ul X ____ml = _____
1 M pNpp (1 ml aliquots @ -20 degrees C) 100 ul X ____ml = _____
1 M Beta-glycerophosphate (4 degrees C) 125 ul X ____ml = _____
2 mM Na3VO4 (1.5 ml aliquots @ -20 degrees C) 250 ul X ____ml = _____
60 mM L-phenylalanine @ 4 degrees C 250 ul X ____ml = _____
B) IP Wash Buffer: (1200 uL X ____samples X ____ IPs + ___ml Bugwash = ____ml)
dH2O/ meq H2O 448 ul X ____ml = _____
5 M NaCl (RT) 50 ul X ____ml = _____
200 mM HEPES (pH 7.3, 4 degrees C) 50 ul X ____ml = _____
5X PIC (on ice from above) 200 ul X ____ml = _____
100 mM Pefabloc-SC (-20 degrees C) 2 ul X ____ml = _____
1% SDS (diluted from 10%, RT) 166.4 ul X ____ml = _____
10% NP-40 (RT) (mix!) 83.2 ul X ____ml = _____ml
Add reagents in order indicated.
Vortex each completely and store on ice throughout use.
On day of Immunoprecipitation:
**Use Program C to cool centrifuge during initial setup.
A) Preparation of SacC Cells:
1. Calculate SacC cells need:
50 ul cells/preclear X ___ reactions + 50 ul X ___ reactions X ___Abs + 10% error factor = ____ml SacC cells.
2. Pellet 5 minutes x 10,000g. Vacuum supernatant.
3. Resuspend in equal volume blocking buffer (1% BSA, 150 mM NaCl, 15mM HEPES, 10 mM EDTA, 5% NP-40, 0.05% NaN3).
4. Incubate on ice 30 minutes (to allow nonspecific site binding).
5. Pellet cells 4 minutes x 3500g. Vacuum supernatant.
6. Resuspend in equal volume IP wash buffer.
7. Repeat 3 times, with final (4th) resuspension in 90% total volume (to account for losses.)
8. Vortex and store on ice throughout use.
B) Preparation of samples:
1. Thaw 50 ul aliquoted samples and add following reagents in order:
**Put aliquots into screw capeppendorf tubes to take intohoodroom (take a little more than is needed).
__ 132 ul dH20
__ 60 ul 5X PIC
__ 15 ul 5 M NaCl
__ 15 ul 200 mM HEPES (pH 7.3)
__ 3 ul 100 mM Pefabloc-SC
__ 25 ul 10% NP-40
(Vortex, pop spin, store on ice)
2. Add 50 ul treated Sacc cells to each tube, vortex, and shake in Eppendorf tube rack on blot rocker in coldroom for 20 minutes (preclearing of SacC cells to eliminate nonspecific binding).
3. Pellet cells 4 minutes x 16,000g.
4. Remove supernatants into another labeled tube (autoclaved screw cap tubes if samples are radioactive) and add antibody directly to that tube (can put antibody in first.)
___ ul _____ antibody added to each tube (X ___tubes), vortex, popspin.
5. Shake in hoodroom on rack for 60 minutes.
6. Add 50 ul treated Sacc cells and shake 30 minutes in coldroom on rack.
7. Pellet immune complexes 4 minutes x 4000g in coldroom centrifuge.
8. Collect supernatants into another labeled tube and add second antibody:
___ ul ____ antibody added to each tube.
9. Shake in hoodroom on rack for 60 minutes.
10. Add 50ul treated Sacc cells and shake 30 minutes in coldroom on rack.
11. Pellet immune complexes 4 minutes x 4000g in coldroom centrifuge.
12. Save supernatants at -20C in freezer in labeled tubes.
C) With immune complex Sacc cell pellets:
1. Wash three times with 400 uL IP wash buffer (resuspend by trituration 200 ul tips).
2. Pellet each time with 4 minutes x 4000g spin in coldroom and aspirate each wash into liquid waste (radioactive waste if samples are hot). Spin aspirated pellets once more and suck off last bit of liquid.
3. After the third wash aspiration, let the pellets dry in hood room 5 minutes.
4. Add 40 ul 1X fresh sample buffer (can freeze and store samples here).
5. Boil samples (in screw cap tubes or with cap guards) 30 minutes with occasional vortexing.
6. Pellet boiled samples 7 minutes x 16,000g.
7. Store samples in freezer at -20C.
D) SDS gels of IP samples:
Run _ IP sample on gel or run equal amounts of protein as determined by BCA.
Sacc cell preparation
A) Preparation of batch for use in several days of IP reactions:
1. 6.5ml of 10% manufacturer suspension pelleted 10?? minutes x 2800g.
2. Resuspend in 10ml freshly made Bugwash (3% SDS, 10% BME, 50mM Tris-HCl pH 7.5, 150mM NaCl).
3. Boil 5 minutes
4. Cool suspension and pellet 10?? minutes x 2800g.
5. Repeat entire process.
6. Resulting pellet suspended in 6ml TEN buffer (50mM Tris-HCl pH7.5, 2mM EDTA, 150mM NaCl).
7. Pellet 10?minutes x 2800g.
8. Repeat 3 times with final pellet being resuspended in 10% final solution in 6ml TEN buffer.
9. Store at 4C.