The conserved motor domain of human KIFC3 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 75 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.
Protein purity is estimated by scanning densitometry of a coomassie-stained SDS-PAGE gradient gel. Figure 1 shows 10 µg of KC01 protein and purity was determined to be >60%. The total protein in each tube will therefore be approximately 40% greater than the amount shown on the tube. The major contaminant at approximately 30 kD is GST protein. The microtubuleactivated ATPase activity of the KIFC3 motor is not inhibited by this contaminant.
Figure 1. KifC3 Motor Domain protein gel. A 10 µg sample of recombinant KifC3 Motor Domain protein (GST-tagged) was separated on a 4-20% SDS-PAGE gradient gel, along with Mark12 molecular weight markers (Invitrogen).The fusion protein runs at 65 kD on the polyacrylamide gradient gel. Protein quantitation was determined using Advanced Protein assay (Cat.# ADV01)
Biological Activity - Microtubule Activated ATPase Assay
KifC3 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 KifC3 microtubuleactivated ATPase activity for this Lot was 90 nmoles ATP generated per minute per mg of KC01 (Figure 2). The ATPase rate for this Lot using a 10 minute endpoint assay (Kinesin ATPase End Point Assay Kit, Cat.# BK053) was 75 nmoles ATP per minute per mg of KC01. Both of these values are above the guaranteed minimum.
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