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N-terminal tagged fusion recombinant fragment, corresponding to amino acids 1-350 of Human BubR1.
Our Abpromise guarantee covers the use of ab4637 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use at an assay dependent dilution.|
|WB||Use a concentration of 10 µg/ml. Detects a band of approximately 130 kDa (predicted molecular weight: 120 kDa). Abcam recommends using milk as the blocking agent. We would suggest using the following protocol; arrest HeLa cells in mitosis with 60ng/ml of sterile nocodazole. Harvest cells by mechanical shake-off between 12 to 16 hours after drug addition. Pellet cells, wash in PBS, and lyse in RIPA. Centrifuge lysates at 4C/15,000g for 10 minutes. Store supernatants and determine protein concentration. Load 40 to 50ug of total protein per lane.|
Immunofluorescent imaging of an asynchronous cycling population of human cells (U2OS) with ab4637 strikingly confirms the specificity of this antibody. No signal is detected from interphase cells, whereas cells undergoing mitosis accumulate BubR1 at the kinetochores. Image reveals kinetochores at prometaphase, and exactly agrees with numerous published images of BubR1 (see for example Johnson et al J Cell Sci. 117(Pt 8):1577-89 (2004).
IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary
antibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes. All blocking and incubation steps carried out at 37 degrees C.
Immunofluorescence of nocodazole treated HeLa cells with ab4637 staining BubR1 (green). CREST was used to mark centromeres (red) and the DNA is stained with DAPI (blue).
Cells were fixed with PFA 4% and permealized with Triton X-100 0.1%. Blocking was done with 4% BSA. ab4637 was used at a 1:200 dilution in 4% BSA for 1.5h at room temperature. Secondary antibodies were incubated for 45min. Coverslips were then stained with DAPI and mounted with Mowiol mounting media.
ab4637 reveals BubR1 in mitotic cells colocalizing with kinetochores, as expected.
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