Myc tag was immunoprecipitated using 0.5mg CHO overexpressing Stra8 whole cell lysate, 5µg of Mouse monoclonal to Myc tag and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, CHO overexpressing Stra8 whole cell lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab56.

Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

Band: 49KDa; Myc tag

Overlay histogram showing HL60 cells stained with ab56 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab56, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-mouse DyLight^® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.