- PA5-29484 - Invitrogen Antibodies HSPA4 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of HSPA4 was performed by separating 30 µg of various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a HSPA4 Polyclonal Antibody (Product # PA5-29484) at a dilution of 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using HSPA4 Polyclonal Antibody (Product # PA5-29484). Sample (30 µg of whole cell lysate). Lane A: Hep G2 . 7.5% SDS PAGE. HSPA4 Polyclonal Antibody (Product # PA5-29484) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of HSPA4 was achieved by transfecting Hep G2 with HSPA4 specific siRNAs (Silencer® select Product # s6976, s6978). Western blot analysis (Fig. a) was performed using whole cell extracts from the HSPA4 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with HSPA4 Polyclonal Antibody (Product # PA5-29484, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25µg/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 HSPA4.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-HSPA4 Rabbit Polyclonal Antibody (Product # PA5-29484) and a 110kDa band corresponding to HSPA4 was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), HeLa (Lane 2), HEK-293 (Lane 3), A549 (Lane 4), SK-BR-3 (Lane 5), NIH/3T3 (Lane 6), PC-12 (Lane 7), COS-7 (Lane 8), MDCK (Lane 9), Mouse Testis (Lane 10) and Rat Ovary (Lane 11) 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)
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Experimental details: Immunofluorescence analysis of HSPA4 was performed using 70% confluent log phase 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 HSPA4 Polyclonal Antibody (Product # PA5-29484) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 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 localization. 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: Immunohistochemistry (Paraffin) analysis of HSPA4 was performed in paraffin-embedded rat brain tissue using HSPA4 Polyclonal Antibody (Product # PA5-29484) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded NCI-N87 xenograft, using HSP70 4 (Product # PA5-29484) antibody at 1:100 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-29484