- PA5-27151 - Invitrogen Antibodies GRB2 antibody

Submitted references Defactinib attenuates osteoarthritis by inhibiting positive feedback loop between H-type vessels and MSCs in subchondral bone.

Hu Y, Wu H, Xu T, Wang Y, Qin H, Yao Z, Chen P, Xie Y, Ji Z, Yang K, Chai Y, Zhang X, Yu B, Cui Z
Journal of orthopaedic translation 2020 Sep;24:12-22

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of GRB2 using 30 µg of Raji lysate. Samples were loaded onto a 12% SDS-PAGE gel and probed with a GRB2 polyclonal antibody (Product # PA5-27151) at a dilution of 1:1000.

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 Cat # 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 # PA5-27151, 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: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of MCF7 (1), A-431 (2), H1975 (3), PC-3 (4), DU-145 (5), MDA-MB-231 (6), Ramos (7), HEK-293 (8), 3T3-L1 (9), Jurkat (10) and A549 (11). The blots were probed with Goat Anti-GRB2 Polyclonal Antibody (Product # PA5-27151, 1:2000 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)
Main image
Experimental details: Western Blot analysis of GRB2 was performed by separating 30 µg of various whole cell extracts by 12% SDS PAGE. Proteins were transferred to a membrane and probed with a GRB2 Polyclonal Antibody (Product # PA5-27151) at a dilution of 1:1000. A: NIH-3T3, B: BCL-1.

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, AssayIDCRISPR1049656_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 # PA5-27151, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27037, 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)
Main image
Experimental details: Western Blot analysis of GRB2 was performed by separating 30 µg of various whole cell extracts by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a GRB2 Polyclonal Antibody (Product # PA5-27151) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry-Immunofluorescence analysis of GRB2 was performed in SK-N-SH cells fixed in 4% paraformaldehyde at RT for 15 min. Green: GRB2 Polyclonal Antibody (Product # PA5-27151) diluted at 1:500. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry-Immunofluorescence analysis of GRB2 was performed in SK-N-SH cells fixed in 4% paraformaldehyde at RT for 15 min. Green: GRB2 Polyclonal Antibody (Product # PA5-27151) diluted at 1:500. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: GRB2 Polyclonal Antibody detects GRB2 protein at nucleus on human breast carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human breast carcinoma. GRB2 Polyclonal Antibody (Product # PA5-27151) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: GRB2 Polyclonal Antibody detects GRB2 protein at nucleus on human colon carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human colon carcinoma. GRB2 Polyclonal Antibody (Product # PA5-27151) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: GRB2 Polyclonal Antibody detects GRB2 protein at nucleus on human ovarian carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human ovarian carcinoma. GRB2 Polyclonal Antibody (Product # PA5-27151) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: GRB2 Polyclonal Antibody detects GRB2 protein at nucleus on mouse liver by immunohistochemical analysis. Sample: Paraffin-embedded mouse liver. GRB2 Polyclonal Antibody (Product # PA5-27151) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: GRB2 Polyclonal Antibody detects GRB2 protein at nucleus on mouse lung by immunohistochemical analysis. Sample: Paraffin-embedded mouse lung. GRB2 Polyclonal Antibody (Product # PA5-27151) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: GRB2 Polyclonal Antibody detects GRB2 protein at nucleus on rat fore brain by immunohistochemical analysis. Sample: Paraffin-embedded rat fore brain. GRB2 Polyclonal Antibody (Product # PA5-27151) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-27151

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