Recombinant Mouse Cdk1, His-GST tagged

• Cat.No.: CDK1-402M
• Product Overview: Recombinant Mouse Cdk1 (P11440) (Met1-Met297), fused with the N-terminal polyhistidine-tagged GST tag, was produced in Baculovirus-Insect cells.

Specification

• Species: Mouse
• Source: Insect Cells
• Tag: Fc
• Protein Length: 1-297 a.a.
• Predicted N Terminal: Met
• Form: Lyophilized from sterile 20mM Tris, 500mM Nacl, 10% glycerol, pH 8.0.Normally 5 % - 8 % trehalose and mannitol are added as protectants before lyophilization.
• Molecular Mass: The recombinant mouse CDK1/GST chimera consists of 534 amino acids and has a calculated molecular mass of 61.9 kDa. The recombinant protein migrates as an approximately 57 kDa band in SDS-PAGE under reducing conditions.
• Endotoxin: < 1.0 eu per μg of the protein as determined by the LAL method.
• Purity: >85 % as determined by SDS-PAGE
• Stability: Samples are stable for up to twelve months from date of receipt at -70oC.
• Storage: Store it under sterile conditions at -20oC~-70oC. It is recommended that the protein be aliquoted for optimal storage. Avoid repeated freeze-thaw cycles.
• Reconstitution: It is recommended that sterile water be added to the vial to prepare a stock solution. Centrifuge the vial at 4℃ before opening to recover the entire contents.
• Publications:
  Cyclin B1 is essential for mitosis in mouse embryos, and its nuclear export sets the time for mitosis (2018)

Gene Information

• Gene Name: Cdk1 cyclin-dependent kinase 1 [ Mus musculus ]
• Official Symbol: Cdk1
• Gene ID: 12534
• mRNA Refseq: NM_007659
• Protein Refseq: NP_031685

Citation

Cyclin B1 is essential for mitosis in mouse embryos, and its nuclear export sets the time for mitosis

Journal: The Journal of Cell Biology    PubMed ID: 29074707    Data: 2018/1/2

Authors: Bernhard Strauss, Andrew Harrison, Jonathon Pines

Article Snippet:Recombinant mouse His-GST CDK1 and recombinant mouse 10xHis CCNB1Myc (Creative BioMart) were used as protein standards and loaded at 1, 5, 10 ng per lane.. Wild-type mouse embryo samples containing 130 uninjected embryos and 130 embryos injected with CyclinB1–Venus mRNA at the zygote stage were harvested at the two-cell stage, and embryo extracts were prepared using lysis buffer (150 mM KCl, 20 mM Hepes, pH 7.6, 2 mM EGTA, 1.5 mM MgCl 2 , 50 mM NaF, 0.1% NP-40, 10% glycerol, 1 mM Na3VO4, 20 mM β-glycerophosphate, 1 mM dithiothreitol, 10 mM benzamidine HCl, and 25 U/ml Benzonase nuclease [Merck]) supplemented with Protease inhibitor cocktail (P2714; Sigma-Aldrich).Wild-type mouse embryo samples containing 130 uninjected embryos and 130 embryos injected with CyclinB1–Venus mRNA at the zygote stage were harvested at the two-cell stage, and embryo extracts were prepared using lysis buffer (150 mM KCl, 20 mM Hepes, pH 7.6, 2 mM EGTA, 1.5 mM MgCl 2 , 50 mM NaF, 0.1% NP-40, 10% glycerol,..

Low amounts of Cyclin B1 are sufficient to trigger mitosis and the threshold is reached by mid S phase. (A) To establish the threshold of Cyclin B1–Venus that can drive null cells into mitosis, fluorescence intensity was used as a read-out for the amount of protein and measured in individual blastomeres of ?/? embryos; y axis arbitrary intensity units. The threshold concentration to trigger mitosis lies between 100 and 200 units above background intensity, corresponding to ～1/10 of the sampled intensity range ( n = 21 blastomeres from five experiments). (B) Wild-type Cyclin B1–Venus was injected into one blastomere of ?/? embryos at the two-cell stage to rescue the arrest phenotype. Signal intensity was measured at the 8- to 16-cell transition after addition of Wee1 inhibitor. Only injected cells with a Cyclin B1 level above a threshold of ～150 arbitrary units can be forced into mitosis by Wee1 inhibition; n = 17, 1 experiment. Error bars are the SD of the mean. (C–F) To test whether there is a time dependency of the inhibitory phosphorylation of Cdk1 by Wee1 the inhibitor MK1775 was added at different phases of the cell cycle to +/+ and +/? embryos. (C) Inhibitor added during mitosis or G1. Only 30% of cells enter to mitosis. 70% of cells arrest after a variable S phase ( n = 35, six experiments). (D) Inhibitor added during early S phase leads to mitotic entry during mid-S phase in most cases; 80% ( n = 36; six experiments). (E) Inhibitor added during mid-S phase forces cells into mitosis mostly in mid- and late S (together 85%; n = 34, six experiments). (F) Inhibitor added during late S phase drives cells into mitosis mostly with a much shortened G2 (see text for details; n = 24, five experiments). ES, early S phase; LS, late S phase; MS, mid-S phase.

Tethering Cyclin B1–Cdk1 to the plasma membrane perturbs its activation. (A–D) Cdk1 antibody staining in Cyclin B1–RFP–Caax mRNA-injected embryos. (A) No primary control. (B) Uninjected embryo showing correct localization of Cdk1 on the spindle during mitosis (right blastomere) and throughout the cell in interphase (left two blastomeres). (C and D) Representative example sections showing Cdk1 localization in embryos injected with Cyclin B1–RFP–Caax. To test the ability of membrane-bound Cyclin B1 to rescue Cyclin B1 ?/? embryos, synthetic mRNA encoding wild-type Cyclin B1–RFP–Caax was injected into one blastomere at the two-cell stage. Representative sections of live embryos at the 32-/64-cell stage for heterozygote (E) and knockout (F) genotypes show localization of membrane targeted cyclin B1. (G) Cell numbers in the injected and uninjected side were counted at this stage and the great majority (90%) of injected embryos arrested either at the four-cell stage or before ( n = 21, 12 experiments) demonstrating the inability of membrane-bound cyclin B1 to rescue B1 ?/? embryos. (H–K) Embryos injected with Cyclin B1–RFP–Caax mRNA were coinjected with a Cdk1 FRET sensor to show changes in levels of Cdk1 phosphorylation. Although Cyclin B1 +/+ and Cyclin B1 +/? embryos show a normal increase of Cdk1 activation before NEBD and a subsequent decrease (H), Cyclin B1 ?/? embryos remained at randomly fluctuating levels (I). (J and K) To test whether Wee1 inhibition can trigger Cdk1 activation in Cyclin B ?/? embryos with membrane-tethered Cyclin B1, FRET imaging was performed to detect changes in Cdk1 phosphorylation. Representative FRET signal graphs aligned to the time point of Wee1 inhibitor (MK 1775) addition. (J) Cyclin B1 +/+ and Cyclin B1 +/? embryos show a clear response after addition of MK1775 (NEBD marked by squares on the graph). (K) Cyclin B1 ?/? embryos injected with membrane-tethered Cyclin B1 showed no response in Cdk1 phosphorylation. There was no difference in FRET measurements close to or away from the membrane. Bars, 20 μm. Imaging interval in FRET analysis, 8 min.