c-MYC 抗体 (AA 31-80)

• Grow HEK-293 cells (ATCC, CRL-1658) in DMEM+GlutaMAXDMEM+GlutaMAX (Gibco, 10567-014, lot 1922818) supplemented with fetal bovine serum (Gibco, 10500-064, lot 08FO477K) and Pen/Strep (Gibco, 15140-122, lot 1924797) at 37°C and 5% CO₂ to 70% confluency.
• Transfect cells either with a Myc expression plasmid (pcDNA3.1-Myc) or HA- and myc-tagged SPAG4 expression plasmids using EndofectinMax (GeneCopoeia) following the manufacturer´s instructions.
• Grow cells for 24h.
• Lyse cells in SDS-sample buffer and denature total cellular lysates for 5min at 95°C. Subsequently separate them on a denaturing 10% SDS-PAGE (Laemmli 1970).
• Transfer proteins onto 0.2µm Protran membrane (GE Healthcare, 10600004, A10043108) by wet blotting for 1h at 400mA (Towbin et al., 1979).
• Block the membrane in TBST (50mM Tris-HCl, pH7.4, 150mM NaCl, 0.2% Tween 20) containing 5% milk (blocking solution) for 60min at RT.
• Incubate membrane with primary rabbit anti-myc antibody (antibodies-online, ABIN1532205 Lot#424170020 and Lot#210020) diluted 1:500 in blocking solution overnight at 4°C.
• Wash membrane with TBST for 45min at RT.
• Incubate membrane with secondary goat anti-rabbit IgG (H+L), HRP-linked (Jackson Immuno Research, 111-035-003, lot 123450) diluted 1:1000 in blocking solution for 45min at RT.
• Wash membrane 6x for 5min with TBST.
• Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062) and capture images via Chemidoc Imaging System (BioRad).
• Incubate membrane with primary mouse anti-HA-tag antibody (clone 12CA5) diluted 1:500 in blocking solution overnight at 4°C.
• Wash membrane with TBST for 45min at RT.
• Incubate membrane with secondary rabbit anti-mouse IgG, HRP-linked (Sigma A9044, Lot 034M4761) diluted 1:1000 in blocking solution for 45min at RT.
• Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062).

In lysates of HEK-293 cells ectopically expressing recombinant protein with an N-terminal HA- and a C-terminal myc-tag ABIN1532205 did not detect the labeled protein at the expected molecular weight. In contrast, an anti-HA-tag antibody (clone 12CA5) revealed a protein at the expected MW.

• Grow HEK-293 cells (ATCC, CRL-1658) in DMEM+GlutaMAXDMEM+GlutaMAX (Gibco, 10567-014, lot 1922818) supplemented with fetal bovine serum (Gibco, 10500-064, lot 08FO477K) and Pen/Strep (Gibco, 15140-122, lot 1924797) at 37°C and 5% CO2 to 70% confluency.
• Transfect cells either with a Myc expression plasmid (pcDNA3.1-Myc) or HA- and myc-tagged SPAG4 expression plasmids using EndofectinMax (GeneCopoeia) following the manufacturer´s instructions.
• Grow cells for 24h.
• Fix cells in 3.7% paraformaldehyde (in PBS) for 20min at 4°C followed by incubation in 0.3% Triton X-100 for 10min at 4°C.
• Block cells in PBS containing 1% BSA and 0.5% Tween-20 (PBT) for 1h at RT.
• Incubate cells with primary antibody
• • rabbit anti-Myc antibody (antibodies-online, ABIN1532205, lot 424170020 or lot 210020) diluted 1:50 in PBS and mouse anti-HA tag antibody (clone 12CA5) ON at 4°C (images G, H).
  • rabbit anti-Myc antibody (antibodies-online, ABIN1532205, lot 210020 or lot 210020) diluted 1:50 in PBS ON at 4°C (images A, D).
• Wash cells with 50mM Tris-HCl, pH7.4, 150mM NaCl, 0.1% Tween 20 (TBST) for 15min at RT.
• Incubate cells with secondary antibody
• • goat anti-rabbit IgG MFP590 (Mobitec MFP-A1037, lot 3002088) diluted 1:300 in PBS and goat anti-mouse IgGDylight488 (Thermo Scientific, 35503) diluted 1:10000 in PBS for 1h at 37°C (images G, H).
  • F(ab')2-Goat anti-Rabbit IgG (H+L) Alexa Fluor 555 (Life Technologies, A21430, lot 1637266) diluted 1:2000 in PBS for 45min at 37°C (images A, D).
• Counterstain cells with DAPI (Sigma, D-9542).
• Image acquisition on Zeiss LSM 510 confocal microscope and processing using Adobe Photoshop 5.0.

The antibody detects Myc proteins by immunoblotting and by immunocytology. No cross-reaction with the C-terminal Myc-tag.