- PA5-16774 - Invitrogen Antibodies ERBB2 antibody

Steinkamp MP, Low-Nam ST, Yang S, Lidke KA, Lidke DS, Wilson BS
Molecular and cellular biology 2014 Mar;34(6):965-77

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on tissue extract (30 µg lysate) of Mouse Ovary (Lane 1), whole cell extracts (30 µg lysate) of A-431 (Lane 2), HeLa (Lane 3), Hep G2 (Lane 4), MCF7 (Lane 5), MDA-MB-231 (Lane 6), T-47D (Lane 7) and SK-OV-3 (Lane 8). The blots were probed with Anti-HER-2 Rabbit Polyclonal Antibody (Product # PA5-16774, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conj µgate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 138 kDa band corresponding to HER-2 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), 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 overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on tissue extract (30 µg lysate) of Mouse Ovary (Lane 1), whole cell extracts (30 µg lysate) of A-431 (Lane 2), HeLa (Lane 3), Hep G2 (Lane 4), MCF7 (Lane 5), MDA-MB-231 (Lane 6), T-47D (Lane 7) and SK-OV-3 (Lane 8). The blots were probed with Anti-HER-2 Rabbit Polyclonal Antibody (Product # PA5-16774, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conj µgate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 138 kDa band corresponding to HER-2 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), 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 overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Her2 was performed using 70% confluent log phase SK-BR-3 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Her2 Rabbit polyclonal Antibody (Product # PA5-16774) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree Celsius and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membranous and cytoplasmic localization. Panel f represents MDAMB-231 cells as negative controls, showing no Her2 staining. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of HER-2/ErbB2 showing staining in the membrane of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a HER-2/ErbB2 Rabbit Polyclonal Antibody (Product # PA5-16774) diluted in 3% BSA-PBS at a dilution of 1:100 for 1 hour at 37°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

PA5-16774