Q2: What is the protocol for using MyBPf as a Kinase substrate?
A2: Follow the protocol suggested by the manufacturer for the Kinase enzyme used in the reaction. An example protocol is shown below: 2.5 μl 10X Abl Kinase Buffer (NEB) 2.5 μl 2 mM ATP 0.5 μl MyBPf (or 0.25 μg) 1 μl Abl Kinase (100 units, NEB #P6050S) Add water to a final reaction volume of 25 μl and incubate at 37°C for 1 hour.
Q3: How do I set up a MyBPf -trypsin digest for mass spectrometry analysis?
A3: A typical reaction is shown below: 2 μl MyBPf (or 1 μg) 3 μl 1X Trypsin buffer 1 μl Modified Trypsin (0.2mg/ml, TPCK-treated, NEB #P8101S) Add water to a final reaction volume of 30 μl. Incubate at room temperature for 1 hour or overnight at 4°C before Mass Spectrometry.
Q4: How much MyBPf do I coat a 96 well plate for a phosphor-ELISA assay?
A4: 5 μg of MyBPf in 100 μl 1X PBS can be used to coat a 96 well micro-titer plate. Incubate the plate overnight at 4°C.
Q5: What are the advantages of using MyBPf compared to tissue extracted MyBP?
A5: 1. MyBPf is a single isoform 2. MyBPf has no post-translational modification, low background in Kinase or other enzymatic assay 3. MyBPf is of high purity and suitable for mass spectrometry analysis.
