ISG15 Antibody MA5-29371 from Thermo Fisher Scientific

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Western blot was performed using Anti-ISG15 Rabbit Monoclonal Antibody (239) (Product # MA5-29371) and a 17 kDa band corresponding to ISG15 was observed across cell lines tested and increased upon IFN alpha treatment. High molecular weight ISG15 conjugates were also observed upon treatment with IFN alpha. Whole cell extracts (30 µg lysate) of A549 (Lane 1), A549 treated with IFN alpha (1000U/mL for 24h) (Lane 2), A549 treated with IFN alpha (1000U/mL for 48h) (Lane 3), HeLa (Lane 4), HeLa treated with IFN alpha (1000U/mL for 48h) (Lane 5), HeLa treated with IFN alpha (1000U/mL for 24h) (Lane 6) and MCF7 (Lane 7) 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 (1:2,000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
ICC/IF

Immunofluorescence analysis of ISG15 was performed using 70% confluent log phase A549 cells treated with IFN alpha (1000U/mL for 48h). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ISG15 Recombinant Rabbit Monoclonal Antibody (239) (Product # MA5-29371) 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 increased ISG15 expression and localization to nucleus and cytoplasm. Panel e shows untreated cells with lower expression of ISG15. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

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ISG15 Antibody

The ISG15 Antibody from Thermo Fisher Scientific is a Rabbit Monoclonal antibody to ISG15, and ISG15 ubiquitin like modifier. This antibody recognizes Homo Sapiens, and Human antigen. The ISG15 Antibody has been shown to work in the following applications: EIA, Immunoassay, ELISA, Immunocytochemistry, and Western Blot.

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ISG15 Antibody from Thermo Fisher Scientific

Thermo Fisher Scientific

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ISG15 Antibody from Thermo Fisher Scientific

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