DYKDDDDK Tag 抗体

• Grow HEK-293 cells (ATCC, CRL-1573) in DMEM+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 with Flag-tagged FE65 expression plasmid (pcDNA3-FE65-FLAG; a kind gift of Dr. Sascha Weggen, University of Düsseldorf) 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 gel (Laemmli 1970) alongside PageRuler Prestained Protein Ladder (ThermoFisher Scientific, 26616).
• Transfer proteins onto 0.2µm Protran membrane (GE Healthcare, 10600004, A10043108) with a western blotting system for 1h at 100V (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 mouse anti-DYKDDDDK antibody (antibodies-online, ABIN3181074, lot 02R690396) diluted 1:2000 in blocking solution ON at 4°C.
• Wash membrane with TBST for 45min at RT.
• Incubate membrane with secondary goat anti-mouse IgG, HRP-linked (Sigma A9044, lot 034M4761) diluted 1:2500 in blocking solution for 45min at RT.
• Wash membrane 6x 5min each with TBST.
• Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062) and capture images via Chemidoc Imaging System (BioRad).
• Incubate a secondary blot membrane with primary rabbit anti-Flag tag antibody (Sigma F7425, lot 086m4803V) diluted 1:10000 in blocking solution ON at 4°C.
• Wash membrane with TBST for 45min at RT.
• Incubate membrane with secondary anti-rabbit IgG (H+L), HRP-linked (Jackson ImmunoResearch, 111-035-003, lot 123450) diluted 1:10000 in blocking solution for 45min at RT.
• Wash membrane with TBST for 45min at RT.
• Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062); image capture via Chemidoc Imaging System (BioRad).

ABIN3181074 does not detect proteins when diluted 1:5000. It does however detect a protein in the expected molecular mass range at approximately 97kDa as well as two additional proteins when diluted 1:2000. A rabbit anti-Flag tag antibody used as reference detects a protein in the expected molecular mass range as well two additional proteins.

• Grow NIH/3T3 cells (ATCC, CRL-1658) in DMEM+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 with DYKDDDDK-tagged FE65 expression plasmid (pcDNA3-FE65-FLAG; a kind gift of Dr. Sascha Weggen, University of Düsseldorf) 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% bovine serum albumin and 0.5% Tween-20 (PBT) for 1h at RT.
• Incubate cells with primary
• mouse anti- DYKDDDDK antibody (antibodies-online, ABIN3181074, lot 02R690396) diluted 1:2000 in PBS ON at 4°C or
  • rabbit anti-DYKDDDDK tag antibody (Sigma, F7425, Lot 086m4803V) diluted 1:2000 in PBS ON at 4°C.
• Wash cells with TBST (50mM Tris-HCl pH7.4, 150mM NaCl, 0.1% Tween 20) for 15min at RT.
• Incubate cells with secondary
• goat anti-mouse IgG Dylight488 (ThermoFisher Scientific, 35503, lot KA) diluted 1:500 in PBS for 1h at 37°C or
  • goat anti-rabbit IgG MFP590 (Mobitec, MFP-A1037, lot 3002088) diluted 1:500 in PBS for 1h at 37°C.
• Counterstain DNA with DAPI (Sigma, D9542).
• Image acquisition on Zeiss LSM 510 confocal microscope and processing using Adobe Photoshop 5.0.