Anti-beta COP抗体(ab2899)

Immunolocalization of beta COP in NIH-3T3 cells using ab2899

Western blot analysis was performed on membrane enriched extracts (30 µg) of cells. The blots were probed with ab2899 (2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate. A ~107 kDa band corresponding beta COP was observed across tissue and cell lines tested. Known quantity of protein samples were electrophoresed using Novex^® NuPAGE^®4-12 % Bis-Tris gel, XCell SureLock™ Electrophoresis System and Novex^® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane by Pierce™ Power Blotter System. The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

Knockdown of beta COP was achieved by transfecting HeLa with beta COP specific siRNAs. Western blot analysis was performed using Membrane Enriched extracts from the beta COP knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ab2899 at 2ug/ml and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (1:4000 dilution). 

Immunofluorescent analysis of beta COP was performed using 70% confluent log phase L6 (Rat skeletal muscle cell line) cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ab2899 at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor^® 488 conjugate a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade^® Gold Antifade Mountant with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor^® 555 Rhodamine Phalloidin. Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.