Cyclin b1

Manufactured by BD

Sourced in United States

Cyclin B1 is a protein involved in the regulation of the cell cycle. It plays a crucial role in the progression of cells through the G2/M phase transition, which is a critical checkpoint in cell division. Cyclin B1 binds to and activates the Cdk1 (Cyclin-dependent kinase 1) enzyme, forming the mitosis-promoting factor (MPF) complex that drives the cell into mitosis.

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Lab products found in correlation

Enhanced chemiluminescence reagent, by Solarbio (1 mentions) Bicinchoninic acid protein assay kit, by Solarbio (1 mentions) Occludin, by Cell Signaling Technology (1 mentions) Vimentin, by BD (1 mentions) P erk, by Merck Group (1 mentions) Snail, by BD (1 mentions) Gapdh, by Merck Group (1 mentions) N cadherin, by Cell Signaling Technology (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Ripa buffer, by Beyotime (1 mentions) Snail c15d3, by Cell Signaling Technology (1 mentions) Hsp90, by BD (1 mentions) Notch4 h 225, by Santa Cruz Biotechnology (1 mentions) Slug c19g7, by Cell Signaling Technology (1 mentions) Flag m2, by Merck Group (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Ab6046, by Abcam (1 mentions) Fxcycle pi, by Thermo Fisher Scientific (1 mentions) Accuri c6 plus flow cytometer, by BD (1 mentions) Hisprobe hrp conjugate, by Thermo Fisher Scientific (1 mentions)

9 protocols using cyclin b1

Quantifying DNA Damage Response Foci

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Cells grown on glass coverslips were fixed with paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked in PBS, 5% BSA, 0.1% Tween 20, stained with anti γH2AX (Upstate) or anti-53BP1 antibodies (Novus Biologicals, 100-304) and counterstained with DAPI. For cyclin B1 staining cells were permeabilized with 0.5% Triton, blocked in 3% BSA and incubated with cyclin B1 (BD Pharmingen) and 53BP1 antibodies. Coverslips were scored by fluorescence microscopy and digital image acquisition on a Nikon Eclipse E1000 equipped with a DS-U3 CCD camera.
γH2AX and 53BP1 foci were stained by immunofluorescence in CCAR2+/+ and CCAR2−/− cells untreated or treated for 1h with etoposide and then released in drug free medium for the indicated time points. Foci were scored on >100 nuclei by fluorescence microscopy using a 100X magnification objective by two independent operators. Standard deviations were calculated on the mean values of at least three independent experiments. P values were determined by t-student test.

Magni M., Ruscica V., Restelli M., Fontanella E., Buscemi G, & Zannini L. (2015). CCAR2/DBC1 is required for Chk2-dependent KAP1 phosphorylation and repair of DNA damage. Oncotarget, 6(19), 17817-17831.

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Comprehensive Protein Expression Analysis

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Total protein was obtained by a RIPA buffer (Beyotime, Shanghai, P.R. China) and quantified with bicinchoninic acid protein assay kit (Solarbio, Shanghai, P.R. China).6 (link) The same quantity of protein (30 µg per lane) was separated by 10% SDS-PAGE. The proteins were incubated with primary antibodies overnight at 4°C against C14orf159 and GAPDH (1:200 and 1:3,000; Sigma-Aldrich Co.), p-ERK, ERK, p-P90RSK, P90RSK, p-P38, P38, p-P65, P65, p-AKT, AKT, Cyclin A2, Cyclin B1, Cyclin D1, Myc-tag, Vimentin, Snail, Slug (1:1,000; BD Transduction Laboratories, Lexington, KY, USA), E-cadherin, N-cadherin, Zo-1 and Occludin (1:1,000; Cell Signaling Technology, Danvers, MA, USA). After incubating with anti-mouse or anti-rabbit IgG (BD Transduction Laboratories) at 37°C for 2 hours, the membranes were developed with enhanced chemiluminescence reagent (Solarbio).

Zhu Y.M., Li Q., Gao X.Z., Meng X., Sun L.L., Shi Y., Lu E.T, & Zhang Y. (2019). C14orf159 suppresses gastric cancer cells’ invasion and proliferation by inactivating ERK signaling. Cancer Management and Research, 11, 1717-1723.

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Mammary Gland Protein Extraction and Immunoblotting

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Total protein extracts were prepared from mammary gland with RIPA buffer. The extracts (30 mg) were subjected to immunoblot analysis as described previously (33) with antibodies to cyclin E (M-20), to p53 (Pab 240), to Mdm2 (SMP14), to c-Myc (N-262), to b-casein (B-5), to Notch2 (M-20), to Notch3 (M-20), and to Notch4 (H-225), all of which were obtained from Santa Cruz Biotechnology; with those to Aurora A and to cyclin B1 (BD Transduction Laboratories); with that to p63 (4A4, Thermo Fisher Scientific); with those to NICD1, to Snail (C15D3), and to Slug (C19G7), all from Cell Signaling Technology; with that to hemagglutinin (HA; HA11, Babco); and with that to FLAG (M2, Sigma-Aldrich). As a control, each membrane was stripped and then probed with antibodies to Hsp90 (BD Transduction Laboratories) or to GAPDH (Santa Cruz Biotechnology).

Onoyama I., Nakayama S., Shimizu H, & Nakayama K.I. (2020). Loss of Fbxw7 Impairs Development of and Induces Heterogeneous Tumor Formation in the Mouse Mammary Gland. Cancer research, 80(24).

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Cell Cycle Analysis of GFP-Rap1 Expressing HeLa Cells

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HeLa cells transfected to express GFP or GFP-Rap1Q63E were synchronised with 100 ng/ml nocodazole for 16 hr at 37°C. Flavopiridol (5 µM) was added for 10 min immediately prior to lysis in RIPA buffer supplemented with PMSF (0.2 mM), NaF (30 mM), and okadaic acid (100 nM). Lysates were analysed by SDS-PAGE and western blotting. Commercially available antibodies were used to Dab2 (sc-13982; Santa Cruz/Insight Biotech, UK), cyclinB1 (554179; BD Biosciences, UK), cdk1 (ab18; Abcam), β-tubulin (ab6046; Abcam), Ect2 (SC-1005; Santa Cruz).

Kaur S., Fielding A.B., Gassner G., Carter N.J, & Royle S.J. (2014). An unmet actin requirement explains the mitotic inhibition of clathrin-mediated endocytosis. eLife, 3, e00829.

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Cell Cycle Analysis by Flow Cytometry

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Cellular DNA content was measured using FxCycle PI (propidium iodide)/RNAse staining solution (Invitrogen, F10797) according to the manufacturer’s instructions. Cyclin B1 quantification was performed in cells fixed with BD fixation/permeabilization solution (BD, 554714, Becton, Dickinson and Company, Heidelberg, Germany) and stained with Cyclin B1-AF647 (Cell Signaling Technology, 4118). The samples were analysed using a BD Accuri C6 Plus flow cytometer (Becton–Dickinson) applying 535/5 nm excitation and emission collected in a 617/20 band-pass. Cells were gated according to their size and granularity; only morphologically intact host cells were included in the analysis. All analyses were performed in FlowJo v.10 software.

Rojas-Baron L., Senk K., Hermosilla C., Taubert A, & Velásquez Z.D. (2024). Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin. Parasites & Vectors, 17, 180.

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Immunoblotting and Immunofluorescence Protocol

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Antibodies against ACA (HCT-0100, ImmunoVision, 1:5000), Aurora B (611082, BD Transduction Laboratories, WB: 1:1000, IF: 1:250), CDH1 (CC43, Millipore, 1:500), Cyclin B1 (554177, BD Pharmingen, 1:5000), EGFP (sc-9996, Santa Cruz, 1:2000), Flag (F1804, Sigma-Aldrich, 1:1000), FOXM1 (sc-500, Santa Cruz, 1:500 for WB; GTX-102170, GeneTex, 1:250 for ChIP), GAPDH (2118, Cell Signaling, 1:5000), HA (home-made, WB: 1:5000), ubiquitin-HRP (AUB01, Cytoskeleton, 1:1000), H3S10ph (9706, Cell Signaling for IF, 1:1000; 06-570, Upstate-Merck, for WB, 1:1000), and RepoMan (HPA030049, Sigma, WB: 1:1000, IF: 1:300) were obtained from the indicated sources. For detection of His-tagged ubiquitin in Supplemental Figures S4G and 5C, the PVDF membrane was incubated with HisProbe-HRP Conjugate (15165, Thermo Scientific, 1: 2500). Secondary HRP–conjugated antibodies were purchased from Dako (Heverlee, Belgium). Secondary Alexa 488, 555, and 633 antibodies were obtained from Invitrogen (Carlsbad, CA).

Manzione M.G., Rombouts J., Steklov M., Pasquali L., Sablina A., Gelens L., Qian J, & Bollen M. (2020). Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells. Molecular Biology of the Cell, 31(6), 419-438.

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Exploring Protein-Protein Interactions

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Pull-down experiments with His-tagged proteins mixed with GST, GST-Survivin or GST-Survivin mutants were carried out as described (78 (link)). For streptavidin-binding experiments, 50 μg of biotinylated peptides were bound to 25–50 μl of a 50:50 slurry of streptavidin agarose (Sigma-Aldrich), mixed with recombinant proteins or cell lysates, and analyzed by Western blotting. Immunoprecipitation using primary antibodies (10 μg/mL), and 200–400 μg of total or fractionated cell extracts were carried out as described (80 (link)). The following antibodies to Cdk1, PT14/PY15-Cdk1, 14–3-3 β, Lamin B (all from Santa Cruz Biotechnology), Cyclin B1 (PharMingen), Survivin (NOVUS Biologicals), α-tubulin (clone B-5–1-2) (Sigma-Aldrich) and His-tag (Invitrogen) were used. Protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences).

, & Cánovas P.M. (2024). Survivin Mediates Mitotic Onset in HeLa Cells Through Activation of the Cdk1-Cdc25B Axis. Research Square.

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Antibody Immunofluorescence Staining Protocol

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Antibodies used in this study were: Mouse monoclonal against dynein intermediate chain clone 74.1 (a gift from Dr. Kevin Pfister), CENP-F (BD Biosciences), p150 (BD Biosciences), cyclin B1 (BD Biosciences), phospho Histone H3 (Abcam), His tag (Genescript), PCNA (Proteintech); Rabbit polyclonal against LIS-1 (Santa Cruz), cyclin B1 (Santa Cruz), PCNA (Abcam), BicD2 (Abcam), BicD2 (a gift from Dr. Anna Akhmanova), RanBP2 (Abcam), NudE/EL (Stehman et al., 2007 (link)); Chicken polyclonal against GFP (Millipore); Rat monoclonal against BrdU (Abcam).

Baffet A.D., Hu D.J, & Vallee R.B. (2015). Cdk1 Activates Pre-Mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem cells. Developmental cell, 33(6), 703-716.

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Western Blot Analysis of Cell Cycle Regulators

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Total cell lysates were subjected to Western blot analysis as described before (9-11). Membranes were probed with antibodies against p21 (mouse monoclonal; 1:400, BD, San Jose, CA, USA), cyclin B1 (mouse monoclonal; 1:350, BD, San Jose, CA, USA), p53 (rabbit polyclonal; 1:300. Cell Signaling, Danvers, MA, USA) or CDK1 (rabbit polyclonal; 1:300 from Cell Signaling Danvers, MA, USA), HPV-18 E2 (rabbit polyclonal Abcam; 1:250, Abcam Cambridge, MA, USA), β-tubulin (rabbit polyclonal Abcam; 1:1000; Cambridge, MA, USA) or calnexin (rabbit polyclonal; 1:500; Novus Biologicals, CO, USA). Primary antibodies were detected using peroxidase-labeled anti-mouse or anti-rabbit IgG (1:5000,). Blots were revealed with the EZ-ECL system (Biological Industries, CT, USA) on a C-DiGit Blot Scanner (LI-COR, Lincoln, NE, USA). The intensity of each band was quantified using ImageJ software (NIH). As positive control for p21 expression, cells were treated with 10 µM H₂O₂ for 48 h [22 (link)].

Villota C., Varas-Godoy M., Jeldes E., Campos A., Villegas J., Borgna V., Burzio L.O, & Burzio V.A. (2018). HPV-18 E2 protein downregulates antisense noncoding mitochondrial RNA-2, delaying replicative senescence of human keratinocytes. Aging (Albany NY), 11(1), 33-47.

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