Blu502z250uc

Manufactured by PerkinElmer

Sourced in United States

The BLU502Z250UC is a laboratory equipment product from PerkinElmer. It is designed to perform a core function within the laboratory environment. The detailed specifications and intended use of this product are not available for an unbiased and factual description at this time.

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Lab products found in correlation

Glutathione sepharose 4 fast flow beads, by GE Healthcare (1 mentions) Histone h2b, by Merck Group (1 mentions) Typhoon instrument, by GE Healthcare (1 mentions)

2 protocols using blu502z250uc

Histone H2B Binding and Phosphorylation Assays

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For H2B and CHMP4B binding assays, GST-CHMP4B (ag4544, Proteintech, Rosemont, IL, USA) was incubated overnight at room temperature with 500 ng of recombinant His-H2B (ag7811, Proteintech) or histone H2B (#14-491, Millipore) in buffer phosphate pH 7.5, 150 mM NaCl. GST-pulldown was performed by incubation for 2 h at 4 °C with Glutathione-Sepharose 4 Fast Flow beads (GE Healthcare, Buckinghamshire, UK) and three washes with buffer phosphate. Bound proteins were resolved by SDS-PAGE and analyzed by WB. For H2B phosphorylation, recombinant His-H2B was incubated with HIPK2 Kinase domain (kind gift of Dr. Linda Montemiglio), as an enzymatic source, in kinase buffer (Hepes 20 mM pH 7.5, 1 mM DTT, 10 mM MgCl2, and 1 mM EGTA) at 30 °C for 30 min in the presence of cold ATP or, as a control, of γ-³²P-ATP (BLU502Z250UC, Perkin-Elmer, Waltham, MA, USA).

Monteonofrio L., Valente D., Rinaldo C, & Soddu S. (2019). Extrachromosomal Histone H2B Contributes to the Formation of the Abscission Site for Cell Division. Cells, 8(11), 1391.

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Phosphorylation Assay for HDHB-EGFP and RbC

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For the in vitro phosphorylation assays with HDHB-EGFP and RbC proteins, substrate concentrations were kept in the range of 1–5 μM. Approximately equal amounts of purified kinase complexes were used, with the exception of Cdk4/cyclin D1, which was about 10-fold in excess compared to other complexes. Reaction aliquots were taken at two time points (8 and 16 min) and the reaction was stopped with SDS-PAGE sample buffer. The basal composition of the assay mixture contained 50 mM HEPES, pH7.4, 180 mM NaCl, 5 mM MgCl₂, 20 mM imidazole. 0.2 mg/ml FLAG peptide, 2% glycerol, 3 mM EGTA, 0.2 mg/ml BSA and 500 uM ATP (with 2 μCi of [γ-32P] ATP added per reaction (PerkinElmer cat# BLU502Z250UC)). To separate the phosphorylated proteins, 10% SDS-PAGE was used for HDHB-EGFP and H1 and 15% SDS-PAGE was used for RbC. Phosphorylation of substrate proteins was visualized using autoradiography (Typhoon instrument; GE Healthcare).

Schwarz C., Johnson A., Kõivomägi M., Zatulovskiy E., Kravitz C.J., Doncic A, & Skotheim J.M. (2018). A Precise Cdk Activity Threshold Determines Passage Through the Restriction Point. Molecular cell, 69(2), 253-264.e5.

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