B-Raf [GST-tagged]

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B-Raf [GST-tagged] kinase is a functional, human, N-terminal GST-tagged, baculovirus/insect cell-expressed, recombinant protein. Identity and activity were confirmed, respectively, by mass spectroscopy and kinase assay using Myelin Basic Protein as a substrate.

Specific activity

54512 Units/mg (45790 Units/ml)

1 Unit = 1 nmole of phosphate incorporated into the substrate (Myelin Basic Protein) in 1 minute

Kinase assay

The specific activity of GST-B-Raf was determined using the method described by Hastie et al. (2006) with the enzyme being assayed at several concentrations. Initially, GST-B-Raf (diluted in 50 mM Tris/HCl pH7.5, 0.1 mM EGTA, 1 mg/ml BSA, 10 mM DTT) was incubated with MKK1 (0.4 μg), p42MAPK (1.4 μg) and ATP (0.1 mM) in 50 mM Tris/HCl pH7.5, 0.1 mM EGTA, 10 mM MgAc, 10 mM DTT buffer for 30 minutes at 30°C. A sample of this GST-B-Raf reaction was then incubated for 10 minutes at 30°C in kinase reaction buffer in the presence of MBP substrate (0.33 mg/ml) and [γ-32P]ATP (100 μM). Duplicate reactions were stopped by spotting the assay mixture onto Whatman P81 paper – capturing the phosphorylated substrate. The radioactivity incorporated was measured on a scintillation counter and the enzyme’s mean specific activity was calculated.

Specifications


• Species:                  human

• Source:                   baculovirus expression vector system

• Quantity:                50 μg

• Concentration:        0.84 mg/ml

• Formulation:          50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 150 mM NaCl, 0.1% β-mercaptoethanol, 270 mM sucrose, 0.03% Brij-35, 1 mM Benzamidine, 0.2 mM PMSF

• Molecular weight:  ~111.8 kDa

• Protein ID:             confirmed by mass spectrometry

• Purity:                    >50% by InstantBlueTM SDS-PAGE

• Stability/Storage:   12 months at -70°C/aliquot as required