- PA5-17692 - Invitrogen Antibodies GRB2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of MCF7 (Lane 1), A-431 (Lane 2), H1975 (Lane 3), PC-3 (Lane 4), DU-145 (Lane 5), MDA-MB-231 (Lane 6), Ramos (Lane 7), HEK-293 (Lane 8), 3T3-L1 (Lane 9), Jurkat (Lane 10) and A549 (Lane 11). The blots were probed with Rabbit Anti-GRB2 Polyclonal Antibody (Product # PA5-17692, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 25 kDa band corresponding to GRB2 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), 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 iBlot® 2 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 Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of GRB2 was achieved by transfecting MCF7 cells with GRB2 specific siRNAs (Silencer® select Product # S6120 and s226232). Western blot analysis (Fig. a) was performed using membrane extracts from the GRB2 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-GRB2 Rabbit Polyclonal Antibody (Product # PA5-17692, 1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to GRB2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing ofGRB2 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR1049656_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of GRB2 was performed by loading 30 µg of HeLa Cas9 (Lane 1) and HeLa Cas9 cells transduced with GRB2 Lentiviral sgRNA (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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 Anti-GRB2 Polyclonal Antibody (Product # PA5-17692, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:5000 dilution).Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). A loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toGRB2 (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of GRB2 was performed using 70% 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 GRB2 Rabbit Polyclonal Antibody(Product # PA5-17692) at 1:100 dilution 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 cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-17692