discontinued

BM4507 BubR1 antibody

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0.1 ml / €390.00

Quick Overview

Mouse anti Human BubR1 P10.8G1.G11.H12

BM4507

Product Description for BubR1

Mouse anti Human BubR1 P10.8G1.G11.H12.
Presentation: Purified
Product is tested for Immunocytochemistry/Immunofluorescence, Immunoprecipitation, Western blot / Immunoblot.

Properties for BubR1

Product Category Primary Antibodies
Quantity 0.1 ml
Synonyms AB4637
Presentation Purified
Reactivity Hu
Applications ICC/IF, IP, WB
Clonality Monoclonal
Clone P10.8G1.G11.H12
Host Mouse
Isotype IgG1
Shipping to Worldwide
PDF datasheet View Datasheet
Manufacturer Acris Antibodies GmbH
Material safety datasheet MSDS for Monoclonal Antibodies (de)

Datasheet Extract

Immunogen
Immunogen:
N-terminal tagged GST fusion recombinant fragment, corresponding to amino acids 1-350 of Human BUBR1
Isotype control AM03095PU-N, SM10P (for use in human samples)
Application Suitable for Immunoprecipitation (3 µg), Immunofluorescence (1/1500) and Western blot (1/500-1/1000, 50 μg of Hela lysate).
Any human cell line can be used as a positive control. Subcellular localization of antigens are only detected in mitotic cells.
Background BUBR1 is a predicted 1050 amino acid mitotic checkpoint kinase which also controls chromosome segregation. It contains 2 domains: CD1 directs kinetochore localization and binding to Bub3, and CD2 contains the kinase domain. Between CD1 and CD2, the BUB1B protein has a putative nuclear localization signal. BUBR1 contains a putative cyclin destruction box that can target proteins for degradation by proteosomes during mitosis.
Concentration 3.0 mg/ml
Protocols Immunofluorescence protocol - Formaldehyde fixation
Collect cells from T.c.unit and remove media from petri dish using suction.
Wash with 1x PBS and remove.
Incubate cells in pre-warm (37°C) Para-Formaldehyde for 12 minutes at room temperature on an orbital shaker.
Remove PFA and incubate in 0.5% Triton X-IOO in 1x PBS for 5 minutes at room temperature.
Prepare blocking reagent, this is also the antibody diluent.
Wash cells 2x with 1x PBS at room temperature, for 4 minutes/wash on an orbital shaker.
Block with 1 % NCS and 1x PBS for 30 minutes at room temperature.
Prepare primary antibodies (50μl/coverslip) and moist staining chambers.
Wash cells 2x with lx PBS at room temperature and air dry briefly.
Incubate with primary antibody for 1 hr at room temperature in the dark in staining chambers. During this time prepare the secondary antibody.
Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)
Incubate with secondary antibody for 1 hour at room temperature in the dark in staining chambers.
Wash cells 5x with 1x PBS.
Mount in Dapi.
Solutions (prepare fresh the same day of staining):
1x Phosphate buffered saline.
Blocking reagent: 1% NCS in 1x PBS (use fresh l0x PBS).
Fixation solution: 3.5% Para formaldehyde.
1.75g PFA in 20 ml d.H20 plus 5 drops 1M NaOH. Stir on a hot plate at 50-60°C until dissolved. Add 4 drops IN HCI and check pH indicator strip. PH should be 7.4. Complete volume with d.H20 to 25ml and add 25ml 2xPBS. Check pH before adding to cover slips.
Immunofluorescence protocol - Methanol/acetone fixation
Collect cells from T.C.unit and remove media from petri dish using suction.
Wash with 1x PBS and remove.
Fix cells with cold methanol: acetone 1: 1 for 10 minutes on ice.
Prepare blocking reagent, this is also the diluent for the antibodies.
Remove fixative and wash cells 3x with Ix PBS at RT, for 4 minutes/wash on orbital shaker.
Block with 1% NCS and Ix PBS for 30 minutes at RT.
Prepare primary antibodies (50μl/coverslip) and moist staining chambers.
Wash cells 2x with 1 x PBS at RT and air dry for approximately 7 minutes.
Incubate with primary antibody for 1 hr at RT in the dark in staining chambers. During this time prepare secondary antibody.
Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)
Incubate with secondary antibody for 1 hr at R T in the dark in staining chambers.
Wash cells 5x with 1x PBS.
Mount in Dapi.
Solutions (prepare fresh the same day of staining):
1x Phosphate buffered saline.
Blocking reagent: 1% NCS in 1x PBS (use fresh 10x PBS).
Fixation solution: methanol:acetone 1: 1 ice cold.
Western Blotting Protocol
Transfer gel to PDVF or nitrocellulose membrane
Place membrane in plastic tray in blocking buffer for one hour with agitation
Rinse in wash buffer
Incubate in wash buffer plus primary antibody for one hour
Wash 6 X 5 minutes with wash buffer
Incubate in wash buffer plus secondary antibody for one hour
Wash 6X 5 minutes with wash buffer
Detect (e.g. ECL, Amersham according to manufacturers instructions)
Wash buffer: PBS + 0.1% Tween 20
Blocking buffer: Wash buffer + 5% dried milk powder
The concentration of antibodies used depends on each antibody, the amount of antigen and the detection method used. Generally, dilution is in the range of a few hundred times dilution to a few thousand times dilution, but usually has to be determined empirically.
General Readings
  1. Zhou J, Panda D, Landen JW, Wilson L, Joshi HC. Minor alteration of microtubule dynamics causes loss of tension across kinetochore pairs and activates the spindle checkpoint. J Biol Chem. 2002 May 10;277(19):17200-8. Epub 2002 Feb 25. PubMed PMID: 11864974.
  2. Liu ST, Hittle JC, Jablonski SA, Campbell MS, Yoda K, Yen TJ. Human CENP-I specifies localization of CENP-F, MAD1 and MAD2 to kinetochores and is essential for mitosis. Nat Cell Biol. 2003 Apr;5(4):341-5. PubMed PMID: 12640463.
Storage Store the antibody at 2-8°C for one month or (in small aliquots) at -20°C for longer.
Avoid repeated freezing and thawing.
Shelf life: One year from despatch.
Format
Purification:
DEAE chromatography.
Buffer System:
PBS with 0.09% sodium azide as preservative.
State:
Liquid purified Ig fraction.
Purified
Specificity
Specificity:
HumanBUBR1.
Not tested for reactivity with rodents or other vertebrates.

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