- 720397 - Invitrogen Antibodies TLK1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of TLK1 was achieved by transfecting PC-3 cells with TLK1 specific validated siRNA (Silencer® select Product # s19124). Western blot analysis (Fig a) was performed using modified whole cell extracts (1% SDS) from the TLK1 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-TLK1 Rabbit Polyclonal Antibody (Product # 720397, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.2 µg/mL, 1:5000 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 TLK1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Modified whole cell extracts (1% SDS) (30 µg lysate) of DU 145 (Lane 1), PC-3 (Lane 2), U-2 OS (Lane 3), T98G (Lane 4), HeLa (Lane 5), NCCIT (Lane 6) and NTERA-2 (Lane 7). The blots were probed with Anti-TLK1 Rabbit Polyclonal Antibody (Product # 720397, 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 87 kDa band corresponding to TLK1 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, cells were fixed and permeabilized for detection of endogenous TLK1 using Anti- TLK1 Rabbit Polyclonal Antibody (Product # 720397, 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 TLK1 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 nuclear localization of TLK1 . Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

720397