- PA5-79523 - Invitrogen Antibodies ISG15 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ISG15 in Lane 1: mouse spleen tissue lysate, Lane 2: mouse heart tissue lysate, Lane 3: mouse lung tissue lysate, Lane 4: mouse liver tissue lysate, Lane 5: mouse kidney tissue lysate using 50 µg (reducing conditions) per well. Electrophoresis was performed on 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours and protein was transferred to a nitrocellulose membrane at 150mA for 50-90 minutes. Sample was blocked with 5% Non-fat Milk/TBS for 1.5 hours at room temperature, incubated with ISG15 polyclonal antibody (Product # PA5-79523) at a dilution of 0.5 µg/mL (overnight at 4°C), followed by goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10,000. Signal development was performed using a chemiluminescence (ECL) kit.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-ISG15 Rabbit Polyclonal Antibody (Product # PA5-79523) and a 17kDa band corresponding to ISG15 was observed across cell lines and tissues tested and increased upon IFN alpha as well as LPS treatment. High molecular weight ISG15 conjugates were also observed upon treatment with LPS. 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), RAW 264.7 (Lane 7), RAW 264.7 treated with LPS (1ug/ml for 24h) (Lane 8), MCF7 (Lane 9) and Mouse Uterus (Lane 10) 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 (0.25ug/ml) 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: Immunohistochemistry analysis of ISG15 on paraffin-embedded mouse kidney tissue. Antigen retrieval was performed using citrate buffer (pH6, epitope retrieval solution) for 20 mins. Sample was blocked using 10% goat serum, incubated with ISG15 polyclonal antibody (Product# PA5-79523) with a dilution of 1 µg/mL (overnight at 4°C), and followed by biotinylated goat anti-rabbit IgG (30 minutes at 37°C). Development was performed using Streptavidin-Biotin-Complex (SABC) with DAB chromogen method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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 Rabbit Polyclonal Antibody (Product # PA5-79523) 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.

PA5-79523