- 701399 - Invitrogen Antibodies ERBB2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ERBB2 was performed by loading 30 µg of MCF7 (lane1), HEK-293 (lane2), Hep G2 (lane3) and SK-Br-3 (lane4) cell lysate using NuPAGE® Novex® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour at room temperature. ERBB2 was detected at ~138 kDa using ERBB2 Recombinant Rabbit Monoclonal Antibody (Product # 701399) at 1-2 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-ErbB2 (HER-2) Recombinant Rabbit Monoclonal Antibody (7H3L20) (Product # 701399) and a ~180kDa band corresponding to Receptor tyrosine-protein kinase erbB-2 was observed across cell lines tested . Whole cell extracts (50 µg lysate) of SK-BR-3 (Lane 1), MCF-7 (Lane 2), SK-O-V3 (Lane 3), Hep G2 (Lane 4), HEK-293 (Lane 5), T-47D (Lane 6), THP-1 (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).Increased expression was observed in SK-BR-3, SKOV-3 and HepG2 as compared to in other cell lines.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HER-2 in whole cell extracts from SK-Br-3 using a HER-2 recombinant rabbit monoclonal antibody (Product # 701399) at a dilution of 1 µg/mL. Detection was performed using an HRP-conjugated goat anti-rabbit secondary antibody followed by chemiluminescence (ECL). Results show a band at ~138kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Receptor tyrosine-protein kinase erbB-2 was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with ErbB2 (HER-2) Recombinant Rabbit Monoclonal Antibody (7H3L20) (Product # 701399) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

701399