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

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.

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.

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.

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).

All lanes : Anti-TRAF2 antibody [EPR6048] (ab126758) at 1/2000 dilution (purified)

Lane 1 : Molt-4 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : HEK293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 55 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

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.

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.

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.

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

Lane 1 : PC12 cell lysate
Lane 2 : MOLT4 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit at 1/2000 dilution

Predicted band size: 55 kDa

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

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

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

Equilibrium disassociation constant (K_D)
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