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The FLAG-HA dual co-immunoprecipitation (co-IP) is an effective procedure for isolating molecular partners of a protein of interest. from a cell population transiently or stably expressing a FLAG- and HA-tagged form of the protein of interest
Materials
1x PBS
Hypotonic Buffer A:
10 mM HEPES pH 7.8, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EGTA, 1 mM PMSF, 1x protease inhibitor cocktail (PIC)
10x Nuclear Isolation Buffer: 150 mM TrisHCl pH 7.4, 150 mM NaCl, 600 mM KCl, 50 mM MgCl2, 1 mM EGTA Sucrose Cushion: 1.2M Sucrose in 1x Nuclear Isolation Buffer supplemented with 1 mM PMSF
Procedure
Wash cells twice with ice-cold PBS
Add 1 mL PBS per plate and harvest by scraping
Spin cells down at 800 x g for 5 minutes at 4°C
Discard supernatant and write down the colume of the cell pellet(packed cell volume, PCV)
Resuspend cells in 5 PCV of hypotonic buffer A and then spin cells down again as before
Discard supernatant and resuspend cells in 4 PCV of hypotonic buffer A
Incubate on ice for 15-20 minutes. Mix samples from time to time by gentle inversion
Transfer cell suspension in a borosilicate Tenbroeck Tissue Grinder and break cells by applying 30 strokes (eventually check cell integrity under the microscope using Trypan Blue solution)
Incubate cell lysate on ice for 10 minutes. Meanwhile, for each sample add 10 PCV of sucrose cushion in a new tube and spin it down at 3000 x g for 5-10 minutes at 4°C
Layer the cell lysate above the sucrose cushion and centrifuge at >5000 x g for 20 minutes at 4°C.
If a large volume of cell lysate is used (> 3 mL), nuclei may get stuck at the interface between ly...