- 700994 - Invitrogen Antibodies EGFR antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of EGFR (Fig a) was performed by loading 30 µg of A-431 Control (lane1), A-431 EGFR knockout (lane 2) whole cell extracts using Novex®NuPAGE®10 % Bis-Tris gel, XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. EGFR was detected at ~ 180 kDa using Recombinant Rabbit Monoclonal EGFR Antibody (Product # 700994, 1 µg/mL) at in 5% skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG (H+L) Recombinant Superclonal™ Secondary Antibody HRP conjugate (Product # A27036, 1:4000 dilution) was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal in CRISPR mediated knockout (KO) confirms that antibody is specific to EGFR.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), HEK-293 (Lane 2), T-47D (Lane 3), HCT116 (Lane 4), A-431 (Lane 5), A549 (Lane 6), U-87 MG (Lane 7) and H1975 (Lane 8). The blot was probed with Recombinant Rabbit Monoclonal EGFR Antibody (Product # 700994, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Recombinant Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 180 kDa band corresponding to EGFR was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE® 10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane using 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 EGFR was performed using 90% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with EGFR Antibody (30H45L42), Recombinant Rabbit Monoclonal (Product # 700994) at 5 microgram/mL in 0.1% BSA, incubated at 4 degree Celsius overnight 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 localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

700994