PINK1 [MBP-tagged]

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PINK1 [MBP-tagged] is a functional, Tribolium castaneum (red flour beetle), N-terminal myelin basic protein-tagged, E. coli-expressed, recombinant protein. Identity was confirmed, by mass spectroscopy and activity by direct and indirect kinase assays.

Kinase activity was determined by two different lot-specific assays:


• Indirect kinase activity assay
   The activity of MBP-PINK1 was validated through its ability to phosphorylate Parkin thus activating and enabling Parkin-catalyzed generation of MBP-PINK1-ubiquitin conjugates. MBP-PINK1 (0, 0.5, 1.0 and 2.0 μg) or MBP-PINK1 (D359A) (0, 0.5, 1.0 and 2.0 μg) were incubated in kinase assay buffer with Parkin (2.0 μg) in the presence or absence of ATP for 60 minutes at 30°C. The ubiquitylation reactions were then initiated through the addition His-UBE1, the E2 conjugating enzyme His-UBE2L3 (UbcH7) and ubiquitin and incubated for a further 60 minutes at 30°C. Elevated MBP-PINK1-ubiquitin conjugates (identified in Western blots with an anti-PINK1 antibody) were observed in reactions with WT as compared to kinase dead (D359A mutant) MBP-PINK1. Ubiquitin conjugates were not observed in the absence of MBP-PINK1 or ATP.

• Direct kinase assay
   MBP-PINK1 (Tribolium castaneum) (diluted in 50 mM Tris/HCl pH 7.5, 10 mM DTT, 0.1 mM EGTA, 1 mg/ml BSA) was assayed in 50μl of 50 mM Tris/HCl pH 7.5, 10 mM DTT, 0.1 mM EGTA, GST-PARK2, 10 mM magnesium acetate, 100 μM [γ-32P]-ATP (50-1000 cpm/pmole), at 30°C for 10 minutes. Reactions were stopped by spotting 40 μl out of the 50 μl assay mixture onto 1.5cm x 1.5cm squares of Whatman P81 paper, sequentially washed in 75 mM phosphoric acid and then acetone before air drying and counting.
   MBP-PINK1 specific activity
   122.13 Units/mg (341.98 Units/ml)
   1 unit = 1nmole of phosphate incorporated into the substrate in 1 minute.


Specifications


• Species:                  Tribolium castaneum (red flour beetle)

• Source:                   E. coli

• Quantity:                50 μg

• Concentration:        1.9 mg/ml

• Formulation:          50 mM Hepes pH 7.5, 150 mM NaCl, 2 mM DTT, 10% glycerol

• Molecular weight:  ~108.1 kDa

• Protein ID:             confirmed by mass spectrometry

• Purity:                    >85% by InstantBlueTM SDS-PAGE

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