KIF18A Motor Domain (1-374) His-Protein: wild-type (Human recombinant)
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.
The wild-type human motor domain of the kinesin-like (KIF18A) 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 1-374. The molecular weight of KIF18A is approximately 44 kDa and it is supplied as a white lyophilized powder.
Protein purity is determined by scanning densitometry of Coomassie Blue-stained protein on a 4-20% polyacrylamide gradient gel. KIF18A 1-374 protein was determined to be 80% pure. (see Figure 1 for an example).
Figure 1 KIF18A 1-374 Protein Purity Determination. A 10 µg sample of recombinant KIF18A protein (molecular weight approx. 44 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.
Microtubule activated ATPase Assay
KIF18A 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 KIF18A micro-tubule-activated ATPase activity is >1500 nmoles ATP generated per minute per mg of KIF18A (See Figure 2 below).
Figure 2: KIF18A microtubule ATPase activity determined with Kinesin ELIPA Kit (Cat#. BK060). Figure legend: KIF18A (0.2 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 3 minutes into the kinetic run. Control reactions (n=5) were carried out in the ab-sence of KIF18A motor protein.