Anti-TRAP1 antibody [TRAP1-6] (ab2721)

ab2721 staining TRAP1 in NCI-H460 cells by Immunocytochemistry/Immunofluorescence. Cells were grown on chamber slides and fixed with formaldehyde. Cells were probed without (right) or primary antibody (left) at a dilution of 1:200 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Green - TRAP1, Red (phalloidin) - F-actin, Blue (DAPI) - nuclei. Images were taken at 60X magnification.

Immunohistochemistry was performed on normal biopsies of deparaffinized Human liver tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer and microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature and probed with a TRAP1 monoclonal antibody (ab2721) at a dilution of 1:20 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

TRAP1 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Mouse monoclonal to TRAP1 (ab2721)and 50µl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10min under agitation. No antibody was added to the control (lane 2). HepG2 whole cell extract diluted in RIPA buffer was added to each sample and incubated for 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab2721. Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Bands: 75kDa: TRAP1