711288

antibody from Invitrogen Antibodies

Targeting: TRAF2 RNF117, TRAP3

Western blot (Supportive data in Antibodypedia)

Antibody Data

Product number

711288

Provider

Invitrogen Antibodies

Product name

TRAF2 Recombinant Polyclonal Antibody (12HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Antibody clone number

12HCLC

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), PC-3 (Lane 2), Jurkat (Lane 3), C2C12 (Lane 4), HEK-293 (Lane 5), THP-1 (Lane 6) and Ramos (Lane 7). The blots were probed with Anti-TRAF2 Recombinant Rabbit Polyclonal Antibody (Product # 711288, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). 61 kDa and 56 kDa bands corresponding to two isoforms of TRAF2 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of TRAF2 was achieved by transfecting HeLa with TRAF2 specific siRNAs (Silencer® select Product # s14379, s14380). Western blot analysis (Fig. a) was performed using whole cell extracts from the TRAF2 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TRAF2 Recombinant Polyclonal Antibody (12HCLC) (Product # 711288, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to TRAF2.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockout of TRAF2 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR856691_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of TRAF2 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) andHeLa TRAF2 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with TRAF2 Recombinant Polyclonal Antibody (12HCLC) (Product # 711288, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to TRAF2.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-TRAF2 Recombinant Polyclonal Antibody (12HCLC), (Product # 711288) and a 55 kDa band corresponding to TRAF2 was observed in the cell lines and tissues tested except in colon tissue which has lower expression. Whole cell extracts (40 µg lysate) of HeLa (Lane 1), THP-1 (Lane 2), Jurkat (Lane 3), HEK-293 (Lane 4), Raji (Lane 5), Mouse thymus (Lane 6), Rat thymus (Lane 7), Mouse colon (Lane 8) and Rat colon (Lane 9) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blots were probed with the primary antibody (0.5 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).