- MA1-19385 - Invitrogen Antibodies CLU antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blotting analysis of human clusterin using mouse monoclonal antibody Hs-3 on lysates of MCF-7 cell line and MOLT-4 cell line (clusterin non-expressing cell line; negative control) under non-reducing and reducing conditions. Nitrocellulose membrane was probed with 2µg/mL of mouse anti-clusterin monoclonal antibody (Product # MA1-19385) followed by IRDye800-conjugated anti-mouse secondary antibody. Clusterin was detected around 40kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Apolipoprotein J was achieved by transfecting HeLa cells with Apolipoprotein J specific siRNAs (Silencer® select Product # s3156, s3158). Western blot analysis (Fig. a) was performed using whole cell extracts from the HeLa knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Apolipoprotein J Mouse Monoclonal Antibody (Product # MA1-19385, 1 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant 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 Apolipoprotein J.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Apolipoprotein J Mouse Monoclonal Antibody (Product # MA1-19385) and 37, 60 kDa bands corresponding to Apolipoprotein J was observed across cell lines tested except LNCaP. An additional band at ~ 38 kDa was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A549 (Lane 2), SW480 (Lane 3), LNCap (Lane 4), PC-3 (Lane 5) and K-562 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 µg/mL) 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 Apolipoprotein J was performed using HeLa 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 Apolipoprotein J Mouse Monoclonal Antibody (Product # MA1-19385) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green) in HeLa cells. 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 merged image of HeLa cells showing cytoplasmic and nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

MA1-19385