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.

Immunoprecipitation of TRAP1 using ab2721 visualized by Coomassie Blue staining.

ICC/IF image of ab2721 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2721, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

All lanes : Anti-TRAP1 antibody [TRAP1-6] (ab2721) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 80 kDa
Observed band size: 76 kDa why is the actual band size different from the predicted?
Additional bands at: 40 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 16 minutes


The band observed at 76 kDa could potentially be a cleaved form of TRAP1 due to the presence of a 59 amino acid transit peptide.

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