KIF19 Motor Domain (1-353) His-Protein: wild-type (Human recombinant)
KIF19 Motor Domain (1-353) His-Protein: wild-type (Human recombinant)
Product uses
• Measurement of microtubule-activated ATPase assays
• Identification/characterization of proteins or small molecules that affect motor ATPase activity.
• Identification/characterization of proteins or small molecules that affect motor/microtubule interactions.
Material
The wild-type human motor domain of the kinesin-like KIF19 (also known as KIF19A) protein has been produced in a bacterial expression system. The recombinant protein contains a 6x His-tag at the amino terminal-end and amino acids 1-353 (Accession number NP_694941.2). The molecular weight of KIF19 is approxi-mately 41 kDa and it is supplied as a white lyophilized powder.
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue-stained protein on a 4-20% polyacrylamide gradient gel. KIF19 1-353 protein was determined to be >85% pure. (see Figure 1).
Figure 1: KIF19 1-353 Protein Purity Determination.
A 10 μg sample of recombinant KIF19 protein (molecular weight approx. 41 kDa) was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue. Protein quantitation was determined using the Precision Red Protein Assay Reagent (Cat. # ADV02). Mark12 molecular weight markers are from Invitrogen.
Biological Activity
Microtubule activated ATPase Assay
KIF19 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 to 360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6- mercapto-7-methylpurine in the presence of inorganic phosphate (Pi). One molecule of Pi will yield one molecule of 2-amino-6- mercapto-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 KIF19 microtubule-activated ATPase activity is >275 nmoles ATP generated per minute per mg of KIF19 (Figure 2).
Figure 2: KIF19 microtubule ATPase activity determined with Kinesin ELIPA Kit (Cat#. BK060). Figure legend: KIF19 (4 µg) ATPase activity was measured (n=5) at an absorbance of 360 nm on an iD5 multi-mode microplate reader (Molecular devices) over 30 minutes at room temperature. The ATPase activity was started when 0.9 mM of ATP was added to the well before starting the kinetic run. Control reactions (n=5) were carried out in the ab-sence of KIF19 motor protein.
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