702322

antibody from Invitrogen Antibodies

Targeting: IRF9 ISGF3G

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

702322

Provider

Invitrogen Antibodies

Product name

IRF9 Recombinant Rabbit Monoclonal Antibody (14H9L22)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Cat, Pig and Horse

Antibody clone number

14H9L22

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Whole cell extracts (30 µg lysate) of Jurakt (Lane 1), Jurkat treated with IFNα (treated with 10 ng/mL for 16 hours) (Lane 2), MCF-7 (Lane 3) and MCF-7 treated with IFNα (treated with 10 ng/mL for 16 hours) (Lane 4). The blots were probed with Anti-IRF9 Recombinant Rabbit Monoclonal Antibody (Product # 702322, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 44 kDa band corresponding to IRF9 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, Ramos cells were fixed and permeabilized for detection of endogenous IRF9 using Anti- IRF9 Recombinant Rabbit Monoclonal Antibody (Product # 702322, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of IRF9 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of IRF9. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Enrichment of endogenous IRF9 protein at specific gene loci using Anti-IRF9 Recombinant Rabbit Monoclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-IRF9 Recombinant Rabbit Monoclonal Antibody (Product # 702322, 5 µg) on sheared chromatin from 2 million Ramos cells treated with 10 nM of human IFN-α1 for 30 min using the MAGnify ChIP system kit (Product # 49-2024). Normal Rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the different loci of the active MX1 promoter region used as positive control target genes, and the region of the inactive SAT2 satellite repeat, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.