Anti-TRAF6 抗体 [EP591Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TRAF6 antibody [EP591Y] (AB33915)

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling TRAF6 with purified ab33915 at 1/240 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRAF6 antibody [EP591Y] (AB33915)

Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using anti-TRAF6 (ab33915)

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TRAF6 antibody [EP591Y] (AB33915)

ab33915 staining TRAF6 in Human platelet cells by intracellular flow cytometry.Cells were fixed in paraformaldehyde and permeabilized using 0.1% Triton-X-100 in 2% BSA for 15 minutes. Primary antibody used at a 1/200 dilution and incubated for 16 hours at 4°C. The secondary antibody used was an Alexa Fluor^®488 conjugated chicken anti-rabbit IgG (H+L) at a 1/500 dilution.

P : permeabilized US : Unstained (Red Peak) IGG RB : IgG Rabbit (Blue Peak) TRAF6 Ab (Green Peak)

This image is courtesy of an anonymous Abreview.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TRAF6 antibody [EP591Y] (AB33915)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-TRAF6 knockout cells (red line) stained with ab33915. The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab33915, 0.1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) preadsorbed (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-TRAF6 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

• WB

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Western blot - Anti-TRAF6 antibody [EP591Y] (AB33915)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : TRAF6 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : HEK293 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab33915 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab33915 was shown to specifically react with TRAF6 in wild-type cells as signal was lost in TRAF6 knockout HAP1 cells. Wild-type and TRAF6 knockout samples were subjected to SDS-PAGE. ab33915 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 2000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging

All lanes:

Western blot - Anti-TRAF6 antibody [EP591Y] (ab33915)

Predicted band size: 60 kDa

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• WB

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Western blot - Anti-TRAF6 antibody [EP591Y] (AB33915)

All lanes:

Western blot - Anti-TRAF6 antibody [EP591Y] (ab33915) at 1/10000 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 60 kDa

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• WB

Lab

Western blot - Anti-TRAF6 antibody [EP591Y] (AB33915)

Lanes 1- 4 : Merged signal (red and green). Green - ab33915 observed at 65 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab33915 was shown to react with TRAF6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab266009 (knockout cell lysate ab257760) was used. Wild-type HeLa and TRAF6 knockout HeLa 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. ab33915 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 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.

All lanes:

Western blot - Anti-TRAF6 antibody [EP591Y] (ab33915) at 1/2000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

TRAF6 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human TRAF6 knockout HeLa cell line (<a href='/products/cell-lines/human-traf6-knockout-hela-cell-line-ab266009'>ab266009</a>)

Lane 3:

HAP1 cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Predicted band size: 60 kDa

Observed band size: 65 kDa

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• WB

Lab

Western blot - Anti-TRAF6 antibody [EP591Y] (AB33915)

All lanes:

Western blot - Anti-TRAF6 antibody [EP591Y] (ab33915) at 1/1000 dilution

Lane 1:

Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 15 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 60 kDa

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• WB

Lab

Western blot - Anti-TRAF6 antibody [EP591Y] (AB33915)

All lanes:

Western blot - Anti-TRAF6 antibody [EP591Y] (ab33915) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

Human heart lysates at 15 µg

Lane 3:

Human skeletal muscle lysates at 15 µg

Lane 4:

Mouse skeletal muscle lysates at 15 µg

Lane 5:

Rat skeletal muscle lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 60 kDa

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