- 702729 - Invitrogen Antibodies ALDOA antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Aldolase A was achieved by transfecting A549 cells with Aldolase A specific siRNA (Silencer® select Product # s72 + s71). Western blot analysis (Fig a) was performed using Whole cell extracts from the Aldolase A knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-Aldolase A Recombinant Rabbit Monoclonal Antibody (Product # 702729, 1-3 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 Aldolase A.

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 A549 (Lane 1), NCI-H1975 (Lane 2), Hep G2 (Lane 3), PANC-1 (Lane 4), SW480 (Lane 5), L6 (Lane 6), C2C12 (Lane 7) and tissue extracts of Mouse Skeletal Muscle (Lane 8). The blots were probed with Anti-Aldolase A Recombinant Rabbit Monoclonal Antibody (Product # 702729, 2.5 µg/mL) 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 40 kDa band corresponding to Aldolase A was observed across the cell lines and tissues tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), 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 iBlot® 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: Knockout of Aldolase A was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR891371_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Aldolase A was performed by loading 30 µg of Hep G2 wild type (Lane 1), Hep G2 Cas9 (Lane 2) andHep G2 Aldolase A KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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-Aldolase A Recombinant Rabbit Monoclonal Antibody (21H7L6) (Product # 702729, 2.5 µg/mL dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:6000 dilution) using the iBright™ FL 1500 (Product # A44115). 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 Aldolase A.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, A549 cells were fixed and permeabilized for detection of endogenous Aldolase using Anti- Aldolase Recombinant Rabbit Monoclonal Antibody (Product # 702729, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Aldolase protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Aldolase A. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

702729