- Proteins and Peptides
- Lysates and Cell Lines
Immunoprecipitation and Western Blotting Procedure
[Buffer recipes to follow protocol]
1. Prepare fresh 1X Lysis buffer (pH 7.4): Need 0.35 ml/pt. + 3 ml/pt.
2. Collect cells by washing them down, trypsinizing or scraping them (as appropriate).
3. Pellet 5 min. @ 1K rpm
4. Remove media.
5. Break pellet by gently tapping it.
6. Wash with X ml cold 1X PBS (pipet up/down once). (X = # of pts. (lanes) to be derived from the cell line.)
7. Aliquot cells 1ml/microfuge tube.
8. Spin cells for 30 seconds @ 12K rpm then place on ice.
9. Aspirate off the supernatant.
10. Lyse cells with 0.35 ml 1X Lysis buffer (added to each tube).
11. Pipet up/down 3-4X to completely resuspend the cells. (**Do not vortex)
12. Incubate for 30 minutes @ 4C, on rotator.
13. Pellet unlysed nuclei down, 5 minutes @ 12K rpm, 4C.
14. Set up anti-TRPM7, NB 500-243 (primary antibody) aliquots into fresh tubes.
15. Transfer the sup into assigned tubes containing the pre-aliquotted primary.
16. Rotate for 2 hours @ 4C on a rotator.
17. Add 0.015 ml of pre-washed Protein-G-Sepharose capturing beads. (Washing protocol provided by bead manufacturer)
18. Rotate 45 minutes @ 4C.
19. Remove from 4C and place on ice.
20. Spin cells for 30 seconds @ 12K rpm.
21. Aspirate off the supernatant.
22. Wash 3x by centrifuging for 1 minute @ 12K rpm and aspirating and resuspending in 1 ml Lysis buffer.
23. Centrifuge for 1 minute @12K rpm, 4 dgrees Celcius.
24. Aspirate off supernatant.
25. Dry pellet with Hamilton syringe (careful not to lose any beads).
26. Resuspend pellet with 0.075 ml (5X stock) reducing SDS-PAGE sample loading buffer.
27. Poke a hole in the top of each tube and boil the samples in a 95 degree Celcius block for 8 minutes.
**At this point the samples are stable indefinitely @ 4C.
28. Quick spin the condensation for 30 seconds @ 12K rpm.
29. Load a 10% gel with the sample.
30. Run the gel overnight at 60V or 6 hours at 140V.
31. Transfer the proteins from the gel to a PVDF membrane (activated by methanol), at 1.4 constant Amp for 3 hours, 20 minutes.
32. Block the membrane in 5% NFDM for >1 hour @ RT, gently shaking.
33. Incubate the membrane with the anti-TRPM7, NB 500-243 overnight @ 4C or for 2 hours @ RT, gently shaking.
34. Wash the blot 5X for 5 minutes, each time, in TBS-T (TBS + 0.05% Tween-20).
35. Incubate the membrane with the anti-rabbit IgG secondary antibody (dilution determined by manufacturer'??s suggestion), diluted in TBS-T + 0.5% NFDM for 45-60 minutes @ RT, gently shaking.
36. Wash the blot 4X for 5 minutes, each time, in TBS-T (TBS + 0.05% Tween-20).
37. Expose the membrane to the ECL reagents of choice.
200 ml 2X Lysis Buffer 20 ml 1X Lysis Buffer
150 mM NaCl 10 ml 2X lysis buffer
80 mM NaF 8.8 ml dH2O
20 mM Iodocetamide 1 ml 10% Triton X-100
100 mM HEPES 0.1 ml PMSF
bring up to 200 ml w/ dH2O 0.1 ml Vanadate
sterile filter, store @ 4 degrees C 111.5 mg Na-pyrophosphate
5X Reducing Sample Loading Buffer
6 ml 1M Tris (pH 6.8)
50 ml 50% Glycerol
10 ml 20% SDS
0.1% (w/v) Bromophenol Blue
5 ml 2-Mercaptoethanol (BME)
Primary Antibody Diluent
* Add BSA to a final concentration of 0.5% before using with the primary antibody