PA5-64093

antibody from Invitrogen Antibodies

Targeting: IRF1 MAR

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-64093

Provider

Invitrogen Antibodies

Product name

IRF1 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Recombinant full-length protein

Description

Immunogen sequence: SAVRVYRMLP PLTKNQRKER KSKSSRDAKS KAKRKSCGDS SPDTFSDGLS SSTLPDDHSS YTVPGYMQDL EVEQALTP

Concentration

0.1 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-IRF1 Polyclonal Antibody (Product # PA5-64093) and a 40 kDa band corresponding to IRF1 was observed across cell lines upon treatment with different interferon inducing agents. Nuclear enriched extracts (30 µg lysate) of MDA-MB-231 (Lane 1), MCF7 (Lane 2), U-937 (Lane 3), U-937 ( treated with PMA/TPA, Human IFN gamma and Lipopolysaccharide) (Lane 4), Hep G2 (Lane 5) and Hep G2 (treated with Human IFN gamma) (Lane 6) 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 (0.5ug/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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of IRF1 was performed using 70% confluent log phase Hep G2 cells treated with Human IFN Gamma (250IU/mL for 24hr). 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 45 minutes at room temperature. The cells were labeled with IRF1 Polyclonal Antibody (Product # PA5-64093) at 1:200 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), (1:3000 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 nuclear localization. Panel e represents no staining in untreated Hep G2 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical staining of IRF1 in human small intestine shows moderate nuclear positivity in glandular cells. Samples were probed using an IRF1 Polyclonal Antibody (Product # PA5-64093).

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Chromatin Immunoprecipitation (ChIP) assay of endogenous IRF1 protein using IRF1 Antibody: ChIP was performed using IRF1 Polyclonal Antibody (Product # PA5-64093, 5 µg) on sheared chromatin from untreated and human IFN Gamma (250IU/mL for 24hr) treated Hep G2 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to MX1, and TRIM22 (Active) and GAPDH (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.