MA1-180

antibody from Invitrogen Antibodies

Targeting: CCNA2 CCN1, CCNA

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

MA1-180

Provider

Invitrogen Antibodies

Product name

Cyclin A2 Monoclonal Antibody (6B4D11)

Antibody type

Monoclonal

Antigen

Other

Description

MA1-180 detects CCNA2 from human and mouse samples.

Antibody clone number

6B4D11

Concentration

1 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), COLO 205 (Lane 2), NIH3T3 (Lane 3), NIH3T3 treated with 0.1 µg/mL nocadazole for 16 hours (Lane 4) and NIH3T3 treated with 0.1 µg/mL of nocodazole for 16 hours and released for 4 hours. The blots were probed with a CCNA2 Mouse Monoclonal Antibody (Product # MA1-180,) at a dilution of 1-2 µg/mL and detected by chemiluminescence using a Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177) at a dilution of 1:2500. A ~49kDa band corresponding to CCNA2 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with the iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary antibody following blocking with 5% Milk in TBST. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of CCNA2 was performed using 70% confluent log phase HeLa cells treated with 0.1 µg/mL of nocodazole for 16 h followed by 4h release. The cell 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 a CCNA2 Mouse Monoclonal Antibody (Product # MA1-180) at a dilution of 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with a Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at 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 (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e is untreated cell with no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.