- 720396 - Invitrogen Antibodies PKN1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (Lane 1), Ramos (Lane 2), A-431 (Lane 3), PC-3 (Lane 4), MDA-MB-231 (Lane 5) and PANC-1 (Lane 6). The blots were probed with Anti-PKN1 Rabbit Polyclonal Antibody (Product # 720396, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.2 µg/mL, 1:5000 dilution). A 130 kDa band corresponding to PKN1 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 Whole cell extracts (30 µg lysate) of U-937 (Lane 1) and U-937 treated with Staurosporine (1µM for 8 hrs) (Lane 2). The blots were probed with Anti-PKN1 Rabbit Polyclonal Antibody (Product # 720396, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.2 µg/mL, 1:5000 dilution). Two isoforms at 130 kDa and 66 kDa corresponding to PKN1 was observed in untreated cells. In treated cells a 50 kDa band corresponding to cleavage product of PKN1 was observed. 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, HeLa cells were fixed and permeabilized for detection of endogenous PKN1 using Anti- PKN1 Rabbit Polyclonal Antibody (Product # 720396, 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 PKN1 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 PKN1 localization in cleavage furrow during cytokinesis. The images were captured at 60X magnification.

720396