Myc tag co ip kit

Antibodies and reagents were obtained from the following sources: the raptor antibody (catalogue No. 09-217) was from Millipore, Schwalbach Germany. mTOR (catalogue No. 2972) and myc-tag (catalogue No. 2272) antibodies were from Cell Signaling Technology, Danvers, MA, USA. The rictor antibody (catalogue No. A300-459A) was from Bethyl Laboratories, Montgomery, TX, USA. hnRNP A2/B1 (catalogue No. sc-32316) and dynamin 2 (catalogue No. sc-6400) was from Santa Cruz Biotechnology, Dallas, TX, USA. Horseradish peroxidase (HRP) labelled secondary antibodies were from Bio-Rad, Munich, Germany. CHAPS buffer was from Applichem, Darmstadt, Germany. The complete protease and phosphatase inhibitor cocktail were from Roche, Mannheim, Germany. Dithiothreitol (DTT), trypsin, trifluoroacetic acid (TFA), formic acid (FA), acetonitrile (ACN) and ammonium bicarbonate (AMBIC) were from Sigma-Aldrich, Steinheim, Germany. RPMI-1640, DMEM, phosphate buffer saline (PBS), penicillin and streptomycin were from PAA Laboratories, Colbe, Germany. The myc-tag Co-IP kit was from Thermo Scientific Pierce, Rockford, IL, USA. The myc-tag raptor pRK5 plasmid was a kind gift of Dr. Doss Sarbassove (The University of Texas, Austin, TX, USA).

CCRF-CEM and HEK293 (8 million each) cells were seeded in 6-well plates for myc-tag raptor pRK5 transfection. Lipofectamine LTX and Plus reagent were used according to the vendor’s recommendations (Invitrogen, Darmstadt, Germany). Briefly, 3 μg of myc-tag raptor pRK5 plasmid and 3 μL of Plus reagent were added to Opti MEM and incubated for five minutes. Then, 4 μL of Lipofectamine LTX were added to the mixture that was then incubated for 30 min at room temperature. The mixture was added to the cells and incubated at 37 °C in a CO₂ incubator for 48 h. Cells were rinsed with cold PBS and lysed on ice-cold CHAPS buffer lacking NaCl to isolate mTOR-containing complexes. Cell lysates were separated from insoluble cell debris by centrifugation at 13,000 rpm for 15 min at 4 °C. A myc-tag Co-IP kit was used according to the manufacturer’s instructions (Thermo Scientific Pierce, Rockford, IL, USA). Briefly, lysates (1 mg/mL) were added to the spin column followed by the addition of myc-tag monoclonal antibody conjugated beads and incubated overnight at 4 °C. Mock controls were run as a negative control. Immunoprecipitates were washed once with CHAPS buffer lacking NaCl and three times with CHAPS buffer containing NaCl and the washes saved. The samples were eluted with glycine buffer (pH 2.8), neutralized by the addition of 1 Mol Tris (pH 9.5) and processed for SDS-PAGE.