Anti-TRAF6 antibody [EP591Y] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TRAF6 antibody [EP591Y] - BSA and Azide free (AB218575)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33915).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRAF6 antibody [EP591Y] - BSA and Azide free (AB218575)

This IHC data was generated using the same anti-TRAF6 antibody clone, EP591Y, in a different buffer formulation (cat# 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)

AbReview22245****

Flow Cytometry (Intracellular) - Anti-TRAF6 antibody [EP591Y] - BSA and Azide free (AB218575)

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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33915).

This image is courtesy of an anonymous Abreview.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TRAF6 antibody [EP591Y] - BSA and Azide free (AB218575)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33915).

• WB

Lab

Western blot - Anti-TRAF6 antibody [EP591Y] - BSA and Azide free (AB218575)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/traf6-antibody-ep591y-ab33915'>ab33915</a>) 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='/en-us/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

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Western blot - Anti-TRAF6 antibody [EP591Y] - BSA and Azide free (AB218575)

This WB data was generated using the same anti-TRAF6 antibody clone, EP591Y, in a different buffer formulation (cat# 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] (<a href='/en-us/products/primary-antibodies/traf6-antibody-ep591y-ab33915'>ab33915</a>)

Predicted band size: 60 kDa

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• WB

CiteAb

Western blot - Anti-TRAF6 antibody [EP591Y] - BSA and Azide free (AB218575)

TRAF6 western blot using anti-TRAF6 antibody [EP591Y] - BSA and Azide free ab218575. Publication image and figure legend from Casella, C. R. & Mitchell, T. C., 2013, PLoS One, PubMed 23638128.

ab218575 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab218575 please see the product overview.

Reduced MyD88-associated signaling by sMLA.Mouse BMDC were treated with 100 ng/ml sMLA or sDLA for the indicated times, lysed and either immunoblots (A,C) or immunoprecipitation (B) were performed on the lysates. A) Immunoblots were probed for IRAK1 and β-actin. A representative gel is depicted with a graph that shows the mean +/- SEM from 5 experiments in which IRAK1 levels were normalized to β-actin levels and vehicle control (VC, measured at 15 min.). B) Lysates were immunoprecipitated with IRAK1 antibodies and immunoblotted with antibodies for TRAF6 and IRAK1. The graph shows mean +/- SEM from 8 experiments with at least 5 data points per time point. TRAF6 levels were normalized based on IRAK1 levels and VC (5 min. time point). C) Left, Immunoblots were probed for phosphoERK 1/2 (Thr202/Tyr204), stripped and re-probed for ERK total, then β-actin. Shown are the mean +/- SEM from 5 experiments with levels of pERK1/2 normalized to ERK total and VC (15 min. time point). Right, immunoblots were probed for IκBα stripped and probed for β-actin. Shown are the mean +/- SEM from 4 experiments with levels of IκBα normalized to β-actin and VC (5 min. time point). Two way ANOVAs with Sidak's multiple comparisons were performed for A and C and two tailed paired T test for B. Asterisks indicate a significant differences between sMLA and sDLA at the indicated time points with * p<0.05, ** p<0.01 and, **** p<0.001, respectively.

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