KIFC3 kinesin motor domain protein: GST tagged: Homo sapiens recombinant

KIFC3 kinesin motor domain protein: GST tagged: Homo sapiens recombinant
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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 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.

Purity
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.

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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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Figure 2. KC01 microtubule-activated ATPase activity using the Kinesin ELIPA Assay Kit (Cat.# BK060).

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Labrière, Christophe et al.New MKLP-2 inhibitors in the paprotrain series: Design, synthesis and biological evaluationsBioorganic and Medicinal Chemistry2016ISSN 1464-3391
Funk, C. Joel et al.Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulatorsAnalytical Biochemistry2004ISSN 0003-2697
Wada, Yuuko et al.Evidence for a Novel Affinity Mechanism of Motor-assisted Transport Along MicrotubulesMolecular Biology of the Cell2000ISSN 1059-1524
Endow, Sharyn A. et al.Determinants of Kinesin Motor PolarityScience1998ISSN 0036-8075

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