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Co-immunoprecipitation (co-IP) is an effective procedure for isolating molecular partners of a protein of interest. The method describes the purification of protein complexes from nuclei of cells stably overexpressing a FLAG-tagged protein of interest. The protocol may be easily adapted for transient transfected cells. DO NOT perform co-immunoprecipitation starting from frozen cell pellets or from frozen cell lysates (freeze-thawing may perturb protein-protein interactions). The whole procedure should be performed within a single day (nuclear extract incubation with M2 anti-FLAG beads may be extended up to O/N). The co-IP protocol may be implemented in a dual co-IP pipeline with the goal of getting IP samples of higher purity when the overexpressed protein has two peptide tags (i.e., FLAG and HA tags)
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
XTR buffer: 50 mM Tris-HCl pH 7.4, 290 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% Glycerol, 0.25% Triton X-100 supplemented with 1 x PIC and 1 mM PMSF, 1 mM NaF, 1 mM NaOV
DIL buffer: 50 mM Tris-HCl pH 7.4, 10 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 0.15% Triton X-100 supplemented with 1 x PIC and 1 mM PMSF, 1 mM NaF, 1 mM NaOV
XTR+DIL buffer: 50% XTR buffer, 50% DIL buffer, supplemented with 1 mM PMSF, 1 mM NaF, 1 mM NaOV
TBS: 50 mM Tris-HCl pH 7.4, 150 mM NaCl
1M GLY: 1 M Glycine pH 2.5
WASH buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Triton X-100, supplemented with 1 mM PMSF
ELU buffer: 150 μg/mL (3x) FLAG peptide in WASH buffer supplemented with 1 x PIC and 1 mM PMSF
SDS-500 buffer: 50 mM Tris-HCl pH 7.4, 500 mM NaCl, 0.15% SDS, supplemented with 1x PIC and 1 mM PMSF
Procedure
Nuclei preparation
Seed cells at a density of 15M cells/plate in 15-cm plates. Use 5 plates per condition. Always include empty vector transfected cells as negative control
Incubate cells at 37°C for 2 days in 25 mL complete medium
Cells should be at a visual confluence of 90% on the day of harvest.
Wash cells twice with 5-10 mL ice-cold PBS
Add 1 mL ice-cold PBS per plate and harvest by scraping. Collect cells in 15 mL tubes (one tube per condition)