- MA5-15348 - Invitrogen Antibodies KRT5 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Cytokeratin (Pan) using a Cytokeratin (Pan) monoclonal antibody (Product # MA5-15348) against a truncated CK5 recombinant protein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Cytokeratin 5 Monoclonal Antibody (7H8C4) (Product # MA5-15348) and a 60 kDa band corresponding to KRT5 was observed across tissues and cell lines tested. Whole cell extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2), A-431 (Lane 3), Mouse Tongue (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Cytokeratin 5 was achieved by transfecting A-431 with Cytokeratin 5 specific siRNAs (Silencer® select Product # s7956, S7957). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Cytokeratin 5 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Cytokeratin 5 Monoclonal Antibody (7H8C4) (Product # MA5-15348, 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 Cytokeratin 5.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Cytokeratin 5 Monoclonal Antibody (7H8C4) (Product # MA5-15348) and a 60kDa band corresponding to Cytokeratin 5 was observed across the cell lines tested except MCF7, IMR-32 and SH-SY5Y along with non specific bands around 30-55 kDa. Whole cell extracts (30 µg lysate) of MCF 10A (Lane 1), MCF7 (Lane 2), A-431 (Lane 3), HaCaT (Lane 4), IMR-32 (Lane 5) and SH-SY5Y (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) 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: Immunofluorescence staining of methanol-fixed Eca-109 (left) and HepG2 (right) cells using Cytokeratin (Pan) monoclonal antibody (Product # MA5-15348)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Cytokeratin 5 was performed using 70% confluent log phase A-431 cells. The cells were fixed with ice-cold acetone at 4°C for 5 minutes and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Cytokeratin 5 Monoclonal Antibody (7H8C4) (Product # MA5-15348) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175), (1:2000 dilution), 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 dilution). Panel d represents the merged image showing cytoskeletal localization. Panel e represents MCF7. 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: Immunohistochemical analysis of paraffin-embedded human lung squamous cell carcinoma (A), normal hepatocyte (B), colon adenocacinoma, normal stomach tissue (D), showing cytoplasmic and membrane localization using Cytokeratin (Pan) monoclonal antibody (Product # MA5-15348) followed with DAB staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded human skin tissues using Cytokeratin (Pan) monoclonal antibody (Product # MA5-15348).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of KRT5 was performed using formalin-fixed paraffin-embedded human skin (malignant melanoma) tissue sections. To expose the target protein, heat-induced epitope retrieval was performed on de-paraffinized sections using eBioscience™ IHC Antigen Retrieval Solution - High pH (10X) (Product # 00-4956-58) diluted to 1X solution in water in a decloaking chamber at 110 degree Celsius for 15 minutes. Following antigen retrieval, the sections were blocked with 2% normal goat serum in 1X PBS for 45 minutes at room temperature and then probed with or without Cytokeratin 5 Monoclonal Antibody (7H8C4) (Product # MA5-15348) at 1:100 dilution in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. Detection was performed using Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) at a dilution of 1:2000 in 0.1% normal goat serum for 45 minutes at room temperature. ReadyProbes™ Tissue Autofluorescence Quenching Kit (Product # R37630) was used to quench autofluorescence from the tissues. Nuclei were stained with DAPI (Product # D1306) and the sections were mounted using ProLong™ Glass Antifade Mountant (Product # P36984). The images were captured on EVOS™ M7000 Imaging System (Product # AMF7000) at 20X magnification and deconvoluted externally.

MA5-15348