- MA5-25362 - Invitrogen Antibodies DNAJB1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of DNAJB1 in human tissue (1: Testis; 2: Omentum; 3: Uterus; 4: Breast; 5: Brain; 6: Liver; 7: Ovary; 8: Thyroid gland; 9: Colon) samples using 10 µg per lane. Samples were separated by SDS-PAGE and probed with DNAJB1 (Product # MA5-25362) monoclonal antibody at a dilution of 1:200.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of DNAJB1 in HEK293T cells in untransfected (Left lane) and transfected (Right lane) samples using 5 µg per lane. The samples were separated by SDS-PAGE and probed with DNAJB1 (Product # MA5-25362) monoclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of DNAJB1 in HeLa cells using 10 µg per lane. Samples were probed with DNAJB1 (Product # MA5-25362) monoclonal antibody at a dilution of 1:200.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of HSP40 was achieved by transfecting HeLa with HSP40 specific siRNAs (Silencer® select Product # s223882, s7009). Western blot analysis (Fig. a) was performed using whole cell extracts from the HSP40 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with HSP40 Monoclonal Antibody (OTI1E9) (Product # MA5-25362, 1:1000 dilution) 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 HSP40.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-HSP40 Monoclonal Antibody (OTI1E9) (Product # MA5-25362) and a 40 kDa band corresponding to HSP40 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), U-87 MG (Lane 2), MCF-7 (Lane 3), Caco-2 (Lane 4), Hep G2 (Lane 5), PC-3 (Lane 6) and A549 (Lane 7) 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 (1:1000 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: Immunofluorescent analysis of DNAJB1 in COS7 cells. Cells were transfected with a plasmid overexpressing DNAJB1 and probed with a DNAJB1 monoclonal antibody (Product # MA5-25362).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of HSP40 was performed using 70% confluent log phase HeLa cells and HeLa cells heat shocked at 43°C for 1 hr. 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 HSP40 Mouse Monoclonal Antibody (OTI1E9) (Product # MA5-25362) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then 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). 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 showing nuclear translocation upon heat shock. Panel e shows untreated cells with weak cytoplasmic signal. Panel f 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 was performed on paraffin-embedded carcinoma of human bladder tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a DNAJB1 monoclonal antibody (Product # MA5-25362).

MA5-25362