Western blot was performed using Anti-POLR1A Polyclonal Antibody (Product # PA5-56766) and a 195 kDa band corresponding to POLR1A was observed along with uncharacterized bands (*) at ~110 , 55 kDa 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) and NTERA-2 (Lane 5) 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 (1:500 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) 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).
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 # PA5-56766) at 1:250 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 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.
Supplier Page from Thermo Fisher Scientific for POLR1A Antibody