- MA5-15707 - Invitrogen Antibodies CSK antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of CSK using CSK monoclonal antibody (Product # MA5-15707) in NIH/3T3 (1) HeLa (2)°COS7 (3), Jurkat (4), Raw246.7 (5), A549 (6), HL-60 (7) and PC-12 (8) cell lysate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of CSK was achieved by transfecting SH-SY5Y cells with CSK specific siRNAs (Silencer® select Product # s3612). Western blot analysis (Fig. a) was performed using whole cell extracts from the SH-SY5Y knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with CSK Monoclonal Antibody (5F3) (Product # MA5-15707, 1:2000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A28177, 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 CSK.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-CSK Monoclonal Antibody (5F3) (Product # MA5-15707) and a 51 kDa band corresponding to CSK was observed across all the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of COLO 205 (Lane 1), HCT 116 (Lane 2), MCF7 (Lane 3), MDA-MB-231 (Lane 4), SK-BR-3 (Lane 5), NIH/3T3 (Lane 6), PC-12 (Lane 7) and tissue extracts of Mouse Liver (Lane 8) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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 (1:1750 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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)
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Experimental details: Immunofluorescence analysis of U251 cells using CSK monoclonal antibody (Product # MA5-15707) (Green). Blue: DRAQ5 fluorescent DNA dye. Red: actin filaments have been labeled with phalloidin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of CSK was performed using 70% confluent log phase HCT 116 cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with CSK Monoclonal Antibody (5F3) (Product # MA5-15707) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) at 1:2000 dilution for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic staining of CSK in HCT 116. 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)
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Experimental details: Flow cytometric analysis of HL-60 cells using CSK monoclonal antibody (Product # MA5-15707) (green) and negative control (purple).

MA5-15707