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Product uses：

Measurement of microtubule-activated ATPase activity

Identification/characterization of proteins or small molecules that affect motor ATPase activity

Identification/characterization of proteins or small molecules that affect kinesin motility

Identification/characterization of proteins or small molecules that affect motor/microtubule interactions

Material：
       The conserved motor domain of human MCAK was expressed in a prokaryotic system. The recombinant protein contains a GST-Tag at the amino terminal end and has a combined molecular weight of 79 kD. The protein has been determined to be biologically active in a microtubule-activated ATPase activity test. The protein is supplied as a lyophilized powder.

Purity：
       Protein purity is estimated by scanning densitometry of a coomassie-stained SDS-PAGE gradient gel. Figure 1 shows 10 μg of MK01 protein and purity was determined to be >80%. The total protein in each tube will therefore be approximately 20% greater than the amount shown on the tube. The major contaminant at approximately 30 kD is GST protein. The microtubuleactivated ATPase activity of the MCAK motor is not inhibited by this contaminant.

       Figure 1. MCAK Motor Domain protein gel. A 10 μg sample of MCAK Motor Domain protein (GST-tagged) was separated on a 4-20% SDSPAGE gradient gel, along with Mark12 molecular weight markers (Invitrogen). The fusion protein runs at 70 kD on the polyacrylamide gradient gel. Protein quantitation was determined using Advanced Protein assay (Cat.# ADV01)

Biological Activity - Microtubule Activated ATPase Assay：
       MCAK ATPase activity was measured by monitoring real time free phosphate generation using the Kinesin ELIPA Assay Kit (Cat.# BK060). The assay is based upon an absorbance shift (330 nm - 360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6mercapto-7-methylpurine in the presence of inorganic phosphate (Pi). One molecule of Pi will yield one molecule of 2-amino-6mercapto-7-methylpurine in an essentially irreversible reaction. Hence, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the kinesin ATPase reaction. Under the conditions outlined below, the Vmax for MCAK microtubuleactivated ATPase activity met or exceeded the minimum activity of 160 nmol/min/mg (Figure 2).

       Figure 2. MCAK microtubule-activated ATPase activity using the Kinesin ELIPA Assay Kit (Cat.# BK060)