Anti-TRAF2 antibody [EPR6048]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TRAF2 antibody [EPR6048] (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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TRAF2 antibody [EPR6048] (AB126758)

Overlay histogram showing HeLa cells stained with unpurifiedab126758 (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRAF2 antibody [EPR6048] (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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TRAF2 antibody [EPR6048] (AB126758)

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TRAF2 antibody [EPR6048] (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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRAF2 antibody [EPR6048] (AB126758)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-TRAF2 antibody [EPR6048] (AB126758)

TRAF2 was immunoprecipitated from HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab126758 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab126758 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate, 10µg Lane 2 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab126758 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Observed MV : 55kDa. Exposure time : 58 seconds.

All lanes:

Immunoprecipitation - Anti-TRAF2 antibody [EPR6048] (ab126758) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab126758 in HeLa whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 55 kDa

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Exposure time: 58s

• WB

Lab

Western blot - Anti-TRAF2 antibody [EPR6048] (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.

All lanes:

Western blot - Anti-TRAF2 antibody [EPR6048] (ab126758)

Predicted band size: 55 kDa

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• WB

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Western blot - Anti-TRAF2 antibody [EPR6048] (AB126758)

All lanes:

Western blot - Anti-TRAF2 antibody [EPR6048] (ab126758) at 1/1000 dilution

Lane 1:

PC12 cell lysate at 10 µg

Lane 2:

MOLT4 cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

293T cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit at 1/2000 dilution

Predicted band size: 55 kDa

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• WB

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Western blot - Anti-TRAF2 antibody [EPR6048] (AB126758)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-TRAF2 antibody [EPR6048] (ab126758) at 1/2000 dilution

Lane 1:

Molt-4 cell lysate at 20 µg

Lane 2:

HeLa cell lysate at 20 µg

Lane 3:

HEK293 cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 55 kDa

Observed band size: 53 kDa

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• WB

Lab

Western blot - Anti-TRAF2 antibody [EPR6048] (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.

All lanes:

Western blot - Anti-TRAF2 antibody [EPR6048] (ab126758) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

TRAF2 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human TRAF2 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-traf2-knockout-hek-293t-cell-line-ab266060'>ab266060</a>)

Predicted band size: 55 kDa

Observed band size: 55 kDa

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• WB

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Western blot - Anti-TRAF2 antibody [EPR6048] (AB126758)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-TRAF2 antibody [EPR6048] (ab126758) at 1/1000 dilution

All lanes:

PC-12 whole cell lysate

Secondary

All lanes:

HRP Goat Anti-Rabbit (H+L) at 1/1000 dilution

Predicted band size: 55 kDa

Observed band size: 53 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-TRAF2 antibody [EPR6048] (AB126758)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• WB

CiteAb

Western blot - Anti-TRAF2 antibody [EPR6048] (AB126758)

TRAF2 western blot using anti-TRAF2 antibody [EPR6048] ab126758. Publication image and figure legend from Zhang, C., Chen, B., et al., 2018, Mol Oncol, PubMed 29377600.

ab126758 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 ab126758 please see the product overview.

TNF‐α/NF‐κB axis is activated in human osteosarcoma cells. (A) The protein levels of critical members in the TNF‐α/NF‐κB axis. The protein levels of TNFR1, TRADD, RIP, TRAF2, IKK, IκBα, phosphorylated IκBα (pIκBα), RelA, RelB, c‐Rel, p50, p52, and CUL4B were measured in hFOB1.19, U2OS, MG63, Saos‐2, and HOS cells. GAPDH was used as the loading control. (B) Effects of SPD304 on the protein levels of the TNF‐α/NF‐κB axis members. hFOB1.19, U2OS, MG63, Saos‐2, and HOS cells were treated with SPD304 for 24 h, and then, the levels of proteins indicated in (A) were determined by immunoblots. (C) Effects of knocking down KNFR1 on the protein levels of the TNF‐α/NF‐κB axis members. hFOB1.19, U2OS, and Saos‐2 cells were transfected with TNFR1‐shRNA, and the stable cell lines were subjected to immunoblots to determine the TNF‐α/NF‐κB axis member protein levels.

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