Anti-TRAP1 抗体 [TRAP1-6]

• WB

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Western blot - Anti-TRAP1 antibody [TRAP1-6] (AB2721)

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.

All lanes:

Western blot - Anti-TRAP1 antibody [TRAP1-6] (ab2721) at 1 µg/mL

Lane 1:

Human brain tissue lysate - total protein (ab29466) at 10 µg

Lane 2:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 80 kDa

Observed band size: 40 kDa,76 kDa

true

Exposure time: 16min

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TRAP1 antibody [TRAP1-6] (AB2721)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRAP1 antibody [TRAP1-6] (AB2721)

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.

• IP

Unknown

Immunoprecipitation - Anti-TRAP1 antibody [TRAP1-6] (AB2721)

Immunoprecipitation of TRAP1 using ab2721 visualized by Coomassie Blue staining.

All lanes:

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

Predicted band size: 80 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-TRAP1 antibody [TRAP1-6] (AB2721)

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

All lanes:

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

Predicted band size: 80 kDa

false

• WB

Unknown

Western blot - Anti-TRAP1 antibody [TRAP1-6] (AB2721)

All lanes:

Western blot - Anti-TRAP1 antibody [TRAP1-6] (ab2721) at 1/2000 dilution

Lane 1:

HeLa whole cell lysate at 30 µg

Lane 2:

Hep G2 whole cell lysate at 30 µg

Lane 3:

HEK-293 whole cell lysate at 30 µg

Lane 4:

K-562 whole cell lysate at 30 µg

Secondary

All lanes:

Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP at 1/40000 dilution

Predicted band size: 80 kDa

false