720297

antibody from Invitrogen Antibodies

Targeting: BUB1B Bub1A, BUBR1, MAD3L, SSK1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

720297

Provider

Invitrogen Antibodies

Product name

BUBR1 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Other

Description

The dual bands between 66-72 kDa detected by this antibody may represent different forms of PRC1.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

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 Hep G2 (Lane 1), MCF7 (Lane 2), PC-3 (Lane 3) and CaCO2 (Lane 4). The blots were probed with Anti-BUBR1 Rabbit Polyclonal Antibody (Product # 720297, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 120 kDa band corresponding to BUBR1 was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and HiMark™ Pre-stained Protein Standard (Product # LC5699). Resolved proteins were then transferred onto a nitrocellulose membrane with overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, Nocodazole (3 uM, 24 h) treated HeLa cells were fixed with methanol and stained with BubR1 Rabbit Polyclonal Antibody (Product # 720297, 2 µg/mL), alpha-Tubulin Monoclonal Antibody (Product # 32-2500, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000), Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor® 594 conjugate (Product # A-11032, 1:400). Panel a) shows representative cells that were stained for detection and localization of BubR1 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal alpha-Tubulin staining (red). Panel d) is a composite image of Panels a, b and c clearly demonstrating punctate nuclear localization for BubR1. Panel e) shows untreated cells with no signal. Panel f) represents control cells with no primary antibody to assess background.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow Cytometry analysis of BUBR1 was performed on HeLa cells and HeLa cells treated with nocodazole (3uM, 24hours) labeled with Anti-BUBR1 Rabbit Polyclonal Antibody (Product# 720297, 5 ug/ 1M cells) or with Rabbit isotype control and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 ug/ml, 1:2500) as represented by the red, blue and pink histograms respectively. The purple histogram represents unstained control cells. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (4468770).