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KIF22 Motor Domain (5-378) His-Protein: wild-type (Human recombinant)

KIF22 Motor Domain (5-378) His-Protein: wild-type (Human recombinant)

$0.00
SKU
CS-KF02
Datasheet
MSDS
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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 KIF22 protein (also known as kinesin-like DNA binding protein, KID) has been produced in a bacterial expression system. The recombinant protein contains a 6x His-tag at the amino terminal-end and amino acids 5-378. The molecular weight of KIF22 is approximately 43 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. Kif22 5-378 protein was determined to be 90% pure. (see Figure 1 for an example).

Click Here for Detailed Methods
cs-kf02

Figure 1. KIF22 5-378 Protein Purity Determination. A 10 µg sample of recombinant KIF22 protein (molecular weight approx. 43 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). Seeblue Pre-stained molecular weight markers are from Invitrogen.

Biological Activity
Microtubule activated ATPase Assay
KIF22 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 KIF22 microtubule-activated ATPase activity is >1200 nmoles ATP generated per minute per mg of KIF22 (Figure 2).

cs-kf02fig2

Figure 2: KIF22 (0.4 µg) ATPase activity was measured (n=4) at an absorbance of 360 nm on an iD5 multi-mode micro-plate reader (Molecular devices) over 30 minutes at room temper-ature. The ATPase activity was started when 0.6 mM of ATP was added 3 minutes into the kinetic run. Control reactions (n=3) were carried out in the absence of KIF22 motor protein.

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