Cytoskeleton, Inc.

Other

Kim Middleton

Ashley Davis

Wai K Law

Li Cheng

Soonjin Hong

Jain Dong Jiang

Henrick Horita

J George Bekesi

The present disclosure provides methods for reducing the viscosity of a cell lysate or tissue lysate by: contacting the cell lysate or tissue lysate with a compressible and open-cell foam filter having a pore size from about 0.65 mm to about 1.22 mm, compressing the filter to recover the lysate absorbed in the filter, and collecting the filtered lysate; and also provides kits therefor.

Methods of reducing viscosity of biological samples

The present invention provides methods of detecting activated Rho GTPase proteins by contacting a solid support with a sample comprising an activated Rho GTPase protein. The solid support is linked to an activated Rho GTPase binding peptide. The activated Rho GTPase protein remains associated with the solid support during the detection.

Detection of Rho proteins

In basic research and drug discovery there is a need to provide protein reagents with high activity. In particular proteins that are used in assays must have a good shelf life and a consistent activity. As a system of measuring actin polymerization, pyrene labeled actin was developed in 1983 by Kouyama and Mihashi. However, prior to this disclosure, pyrene actin has been impossible to store for longer than one month because the activity would be lost due to protein denaturation and frozen pyrene actin immediately lost activity. This disclosure relates a method for stabilizing pyrene labeled actin for extended storage times and an optimal storage buffer thus creating a reproducible source of pyrene actin for various assays. Using the processes described herein, pyrene actin has been stored for greater than 3 years at 4&#xb0; C. which makes it a viable product for retail.

Methods for stabilizing proteins

It is presently accepted that the mechanism of action for all anti-tumor tubulin ligands involves the perturbation of microtubule dynamics during the G2/M phase of cell division and subsequent entry into apoptosis (1). In this invention, we report a novel tubulin ligands which have a unique mechanism of action. These compounds, halogenated derivatives of acetamido benzoyl ethyl ester (HAABE), arrest cancer cells in S-phase and cause cell death by a combination of apoptosis (DNA ladder) and necrosis (DNA degradation) type mechanisms. Normal cells are not affected at the same concentrations of compound. The ligands bind covalently to tubulin in vitro and in vivo and shows potent cancericidal activity in tissue culture assays and in animal tumor models. These compounds target early S-phase at the G1/S transition rather than the G2/M phase and mitotic arrest. Bcl-2 phosphorylation, a marker of mitotic microtubule inhibition by other tubulin ligands was dramatically altered, phosphorylation was rapid and biphasic rather than a slow linear event. The halogenated ethyl ester series of derivatives thus constitute a unique set of tubulin ligands which induce a novel mechanism of cancer cell death.

Anti-S-phase tubulin ligands

Lyophilized active tubulin and numerous uses of lyophilized active tubulin such as diagnostic, therapeutic, drug discovery, and research applications are provided. The process of making the lyophilized active tubulin is also described.

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Denver, CO, United States of America

Kim Middleton