All lanes : Anti-TRAF2 antibody [EPR6048] (ab126758) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : TRAF2 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 55 kDa

This data was developed using the same antibody clone in a different buffer formulation (ab126758).

Lanes 1- 2: Merged signal (red and green). Green - ab126758 observed at 55 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab126758 was shown to react with TRAF2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266060 (knockout cell lysate ab257759) was used. Wild-type HEK-293T and TRAF2 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab126758 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Unpurified ab126758, at 1/50 dilution, staining TRAF2 in paraffin-embedded Human kidney tissue, by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126758).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

This WB data was generated using the same anti-TRAF2 antibody clone, EPR6048, in a different buffer formulation (cat# ab126758).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: TRAF2 knockout HAP1 cell lysate (20 µg) 
Lane 3: Human skeletal muscle lysate (20 µg)
Lane 4: U2OS cell lysate (20 µg) 
Lanes 1 - 4: Merged signal (red and green). Green - ab126758 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab126758 was shown to specifically react with TRAF2 when TRAF2 knockout samples were used. Wild-type and TRAF2 knockout samples were subjected to SDS-PAGE. ab126758 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

ab126758 (purified) at 1/40 immunoprecipitating TRAF2 in HEK293 (Lane 1 and 2). Lane 3 - PBS. For western blotting a HRP-conjugated anti-rabbit IgG specific to the non-reduced form of IgG was used as the secondary antibody (1/1500). Blocking buffer and concentration: 5% NFDM/TBST. Diluting buffer and concentration: 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126758).

Overlay histogram showing HeLa cells fixed in 2% PFA and stained with purified ab126758 at a dilution of 1 in 120 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 150. Rabbit monoclonal IgG was used as an isotype control (black) and cells without antibody were used as a negative control (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126758).

Immunofluorescence staining of HeLa cells with purified ab126758 at a working dilution of 1 in 100, counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab126758 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126758).

Overlay histogram showing HeLa cells stained with unpurified ab126758 (red line). The cells were fixed with 80% methanol (5 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 (unpurified ab126758, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126758).

Unpurified ab126758, at 1/100 dilution, staining TRAF2 in HeLa cells, by Immunofluorescence.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126758).

Equilibrium disassociation constant (K_D)
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This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126758).

This IHC data was generated using the same anti-TRAF2 antibody clone, EPR6048, in a different buffer formulation (cat# ab126758).

Immunohistochemical staining of paraffin embedded human cervical cancer with purified ab126758 at a working dilution of 1/100. The secondary antibody used is ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.