Hoatlin:IP Protocol

HOATLIN LAB IP PROTOCOL

LYSIS BUFFER 10mM Tris pH 7.4 150mM NaCl 1% NP-40 0.5% Sodium Deoxycholate 1mM EDTA 1mM DTT 0.5mg/ml pefabloc protease inhibitor (Boeringer) --> Freeze aliquots at –80 for future use. Keep lysis buffer on ice when in use.

IP (for transfected cells)  -*500,000 (Transfected) cells in 500ul lysis buffer + 5uL normal serum, rock 90min. @4C. -Add 50uL Protein A/G beads, rock 30min @ 4C. -Spin 15sec. @ 4C, 13,000 rpm -Recover Supernatant. Add to supernatant 5 uL serum or 1 ug purified antibody, rock @ 4C, 2-16 hours. (best results O/N) -Add 30uL protein A/G beads, rock 30min @ 4C. -Wash beads 3 X 30sec inversion with 500-100ul cold lysis buffer. -Spin beads between each wash -Add 20uL 2X sample buffer, boil 5-10min, ice 2min. Load 15uL on gel.

IP (for endogenous FA proteins) -*20 million cells in 1mL lysis buffer + 5uL normal serum, rock 90min. @4C. -Add 50uL Protein A/G beads, rock 30min @ 4C. -Spin 15sec. @ 4C, 13,000 rpm -Recover Supernatant. Add to supernatant 5 uL serum or 1 ug purified antibody, rock @ 4C, 2-16 hours. (best results O/N) -Add 50uL protein A/G beads, rock 30min @ 4C. -Wash beads 3 X 30sec inversion with 500-100ul cold lysis buffer. -Spin beads between each wash -Add 20uL 2X sample buffer, boil 5-10min, ice 2min. Load 15uL on gel.

(*) After adding lysis buffer sheer cells with 19 gauge needle 5X, spin out and discard cell pellet.

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