702748

antibody from Invitrogen Antibodies

Targeting: PCK2 PEPCK, PEPCK2

Antibody data

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Validation data

Product number: 702748 - Provider product page
Provider: Invitrogen Antibodies
Product name: PCK2 Recombinant Rabbit Monoclonal Antibody (16H5L22)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: This antibody is predicted to react with Monkey, Rabbit, Bovine, Horse
Antibody clone number: 16H5L22
Concentration: 0.5 mg/mL

PCK2 protein structure - 702748 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of PCK2 was achieved by transfecting A549 cells with PCK2 specific siRNA (Silencer® select Product # s10126 + s10125). Western blot analysis (Fig a) was performed using Membrane enriched extracts from PCK2 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-PCK2 Recombinant Rabbit Monoclonal Antibody (Product # 702748, 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 PCK2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Membrane enriched extracts (30 µg lysate) of A549 (Lane 1), Hep G2 (Lane 2), HT-29 (Lane 3), MDA-MB-231 (Lane 4) and Jurkat (Lane 5). The blots were probed with Anti-PCK2 Recombinant Rabbit Polyclonal Antibody (Product # 702748, 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 70 kDa band corresponding to PCK2 was observed across the cell lines 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)
Main image
Experimental details: Western blot analysis was performed on Membrane enriched extracts (30 µg lysate) of A549 (Lane 1), Hep G2 (Lane 2), HT-29 (Lane 3), MDA-MB-231 (Lane 4) and Jurkat (Lane 5). The blots were probed with Anti-PCK2 Recombinant Rabbit Polyclonal Antibody (Product # 702748, 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 70 kDa band corresponding to PCK2 was observed across the cell lines 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)
Main image
Experimental details: For immunofluorescence analysis, HepG2 cells were fixed and permeabilized for detection of endogenous PCK2 using Anti- PCK2 Recombinant Rabbit Monoclonal Antibody (Product # 702748, 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 PCK2 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 Mitochondrial localization of PCK2. 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)
Main image
Experimental details: For immunofluorescence analysis, HepG2 cells were fixed and permeabilized for detection of endogenous PCK2 using Anti- PCK2 Recombinant Rabbit Monoclonal Antibody (Product # 702748, 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 PCK2 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents mitochondrial staining using MitoTracker® Red CMXRos (Product # M7512). Panel d) is a composite image of Panels a, b and c clearly demonstrating co-localization of PCK2 with mitotracker. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.