Hoatlin:IP Protocol

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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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