Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

SignalSilence® Bub1b siRNA II #12150

Applications Reactivity
Transfection H (Mk)

Reactivity Key:  H=Human  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® Bub1b siRNA I #6623 (+), or SignalSilence® Bub1b siRNA II (+), using Bub1b (D32E8) Rabbit mAb #5421 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The Bub1b (D32E8) Rabbit mAb confirms silencing of Bub1b expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

Description

SignalSilence® Bub1b siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Bub1b expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

Quality Control

Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.

Directions for Use

CST recommends transfection with 100 nM SignalSilence® Bub1b siRNA II 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use.

Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.

Background

The Mitotic Checkpoint Complex (MCC), which contains Bub1, Bub1b, Bub3, Mad2, and Cdc20, controls chromosome segregation and monitors kinetochore-microtubule interactions (1). During mitosis, the MCC complex inhibits the ubiquitin ligase activity of the Anaphase Promoting Complex/Cyclosome (APC/C), thereby preventing cells with unaligned chromosomes from prematurely entering anaphase (2). Research studies have shown that Bub1b and Bub1 kinases are mutated in several types of human malignancies including hematopoietic, colorectal, lung, and breast cancers (3). Biallelic mutations in Bub1b have been found in mosaic variegated aneuploidy syndrome and premature chromatid separation syndrome (4). Bub1b mouse germline knockouts are embryonic lethal with heterozygous animals displaying genetic instability, early aging phenotypes, and increased cancer susceptibility (5). Bub3 binds both Bub1 and Bub1b, facilitating their recruitment to kinetochores (6), and is required for functional microtubule-kinetochore interactions (7).

  1. Fukagawa, T. (2008) Front Biosci 13, 2705-13.
  2. Chen, R.H. (2007) J Biomed Sci 14, 475-9.
  3. Dai, W. et al. (2004) Cancer Res 64, 440-5.
  4. Kops, G.J. et al. (2005) Nat Rev Cancer 5, 773-85.
  5. Baker, D.J. et al. (2004) Nat Genet 36, 744-9.
  6. Taylor, S.S. et al. (1998) J Cell Biol 142, 1-11.
  7. Logarinho, E. et al. (2008) Mol Biol Cell 19, 1798-813.

Application References

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Companion Products

Limited Use Label License, RNA interference: This product is licensed under European Patent 1144623 and foreign equivalents from Ribopharma AG, Kulmbach, Germany and is provided only for use in non-commercial research specifically excluding use (a) in drug discovery or drug development, including target identification or target validation, by or on behalf of a commercial entity, (b) for contract research or commercial screening services, (c) for the production or manufacture of siRNA-related products for sale, or (d) for the generation of commercial databases for sale to Third Parties. Information about licenses for these and other commercial uses is available from Ribopharma AG, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Germany.

For Research Use Only. Not For Use In Diagnostic Procedures.

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