- MA5-14874 - Invitrogen Antibodies PRKCI antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of PKC-lambda in extracts from C2C12 and PC12 cells using PKC-lambda monoclonal antibody (Product # MA5-14874).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of PRKCI was achieved by transfecting HeLa with PRKCI siRNAs (Silencer® select Product # s71706, s71707). Western blot analysis (Fig. a) was performed using whole cell extracts from the PRKCI knockdown cells (lane 2) and non-specific scrambled siRNA transfected cells (lane 1). The blot was probed with PRKCI Monoclonal antibody (Product # MA5-14874, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25 µg/mL, 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 PRKCI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-PRKCI Antibody (Product # MA5-14874) and a 78 kDa band corresponding to PRKCI was observed across the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of PC-12 (Lane 1), C2C12 (Lane 2), Mouse Lung (Lane 3) and Rat Lung (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of PRKCI was performed using C2C12 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with PRKCI Rabbit Monoclonal Antibody (Product # MA5-14874) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32790) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the composite image showing cytoplasmic staining of PRKCI in C2C12 cells. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

MA5-14874