PA5-65577

antibody from Invitrogen Antibodies

Targeting: POLR1A DKFZP586M0122, FLJ21915, RPA1, RPA190, RPO1-4

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-65577

Provider

Invitrogen Antibodies

Product name

POLR1A Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Recombinant full-length protein

Description

Immunogen sequence: DEVRGKWQDAH LGKDQRDFNM IDLKFKEEVN HYSNEINKAC MPFGLHRQFP ENSLQMMVQS GAKGSTVNTM QISCLLGQIE LEGR

Concentration

0.2 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-POLR1A Polyclonal Antibody (Product # PA5-65577) and a 195 kDa band corresponding to POLR1A was observed along with uncharacterized bands (*) across cell lines tested. Whole cell Extracts (1% SDS) (30 µg lysate) of K-562 (Lane 1), Caco-2 (Lane 2), A549 (Lane 3), HeLa (Lane 4), NTERA-2 (Lane 5), MCF-7 (Lane 6) and RAW 264.7 (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by wet transfer XCell SureLock™ Mini-Cell and XCell II™ Blot Module (Product # EI0002). The blot was probed with the primary antibody (1µg/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

Immunofluorescent staining of POLR1A in human cell line U-2 OS shows localization to nucleus and nucleoli fibrillar center. Samples were probed using a POLR1A Polyclonal Antibody (Product # PA5-65577).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of POLR1A was performed using 70% confluent log phase A-431 cells. 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 1 hour at room temperature. The cells were labeled with POLR1A Polyclonal Antibody (Product # PA5-65577) at 5 µg/mL 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 ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415). Panel d represents the merged image showing Nucleolar localization. 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

Chromatin Immunoprecipitation (ChIP) assay of endogenous POLR1A protein using Anti-POLR1A Antibody: ChIP was performed using Anti-POLR1A Rabbit Polyclonal Antibody (Product # PA5-65577, 5 µg) on sheared chromatin from HeLa 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 28s rDNA transcriptional start site and gene body, 5s rDNA transcriptional start site and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.