- MA1-24952 - Invitrogen Antibodies PXN antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), NIH/3T3 (Lane 3), Panc-1 (Lane 4), A549 (Lane 5) and HT-1080 (Lane 6). The blot was probed with Anti-Paxillin Monoclonal Antibody (Product # MA1-24952, 1:500 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/ml, 1:4000 dilution). Multiple bands ranging from 44-65 kDa corresponding to different isoforms of Paxillin was detected across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of Paxillin was performed by loading (1) HEK-293T (2) HeLa (3) A431 (4) JURKAT (5) NIH-3T3 cell lysates. Proteins were transferred to a membrane and probed with a Paxillin Monoclonal Antibody (PXC-10) (Product # MA1-24952) at a dilution of 1:500..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Paxillin was achieved by transfecting PANC-1 cells with Paxillin specific siRNAs (Silencer® select Product # s11627, s11629). Western blot analysis (Fig. a) was performed using whole cell extracts from the Paxillin knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Paxillin Monoclonal Antibody (PXC10) (Product # MA1-24952, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Paxillin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of Paxillin was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR1071991_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Paxillin was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2), HeLa Paxillin KO (Lane 3) whole cell extracts. The samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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-Paxillin Monoclonal Antibody (PXC-10)(Product # MA1-24952) using 1:500 dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Paxillin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Paxillin was performed using 70% confluent log phase HeLa 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 Paxillin Rabbit Polyclonal Antibody (Product # MA1-24952) at 1:250 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) 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 membrane and cytoplasmic localization. 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)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of Paxillin in HeLa cells using Paxillin Monoclonal Antibody (PXC-10) (Product # MA1-24952) at 1:200 (red). Cells were fixed and permeabilized with 4% PFA followed by 0.5% Triton X-100.

MA1-24952