Anti c myc antibody

COS‐7 cells were cultured in RPMI supplemented with 10% FCS and 100 μg/mL pen/strep. For expression experiments, approximately 1 × 10⁶ cells were seeded into 100 mm dishes and transfected at 60–80% confluency. Using Lipofectamine™ (Invitrogen), 6 μg of each construct was cotransfected with a control plasmid (4 μg). Each transfection was performed at least in triplicate and with two different DNA preparations of each construct. Cells were harvested 24 h posttransfection, lysed in 300 μL RIPA buffer, sonified and centrifuged at 10000 rpm for 1 min. Cell lysates were resolved by SDS‐PAGE on a 12% acrylamide gel and analyzed by immunoblotting using primary mouse anti‐c‐myc‐antibody (1:1000, BD Bioscience, Heidelberg, Germany) at 4°C over night, followed by a secondary antibody (1:5000) for 1 h at RT. The signals were monitored by incubating with ECL reagent (Amersham) for 1 min. A Fuji Medical Super RX X ray film (Fuji, Düsseldorf, Germany) was exposed according to the signal intensity.

500 μg of total protein from the cell extracts were incubated overnight at 4° C with anti-c-Myc antibody (BD Pharmingen, 551101). 50 μL of Sepharose-G beads were added and incubated for 2 hours at 4° C. Immunoprecipitated complexes were denatured with SDS-PAGE sample buffer (90° C for 5 min), separated by SDS-PAGE and analyzed by immunoblotting as previously described [26 (link)] using as primary antibodies anti-GFP from Developmental Studies Hybridoma Bank (DHSB GFP-G1) 1:1000 dilution and c-Myc (BD Pharmingen, 551101) 1:1000 dilution. Secondary antibody (Santa Cruz, anti-mouse sc-2031) 1:3000 dilution was incubated 1 hour at room temperature. Detection was carried out with Illumina Crescendo reagent (Millipore).