Western blot was performed using Anti-Dynactin 1 Polyclonal Antibody (Product # PA5-21289) and a 141 kDa band corresponding to DCTN1 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of Jurkat (Lane 1), A-431 (Lane 2), MCF7 (Lane 3), COS-7 (Lane 4), Hep G2 (Lane 5) and HeLa (Lane 6) were electrophoresed using NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1500 dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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 Dynactin 1 was performed using 70% confluent log phase HeLa 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 Dynactin 1 Polyclonal Antibody (Product # PA5-21289) 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™ 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, 1:300). Panel d represents the merged image showing cytoskeleton localization. Panel e represents cells with no primary antibody to assess background. The images were captured at 60X magnification..
Supplier Page from Thermo Fisher Scientific for Dynactin 1 Antibody