Compound Screening, Protein Purification, & Assay Development

As Cytoskeleton’s Custom Services Department continues to grow and expand its offerings, we wanted to take the opportunity to highlight some past research projects that benefited from work performed by Cytoskeleton’s Custom Services scientists. The three main foci of Cytoskeleton’s Custom Services are: 1) Compound Screening, 2) Assay Development, and 3) Gene Cloning and Recombinant/Native Protein Purification. The citations discussed below demonstrate our effectiveness in all three types of custom services.

Compound Screening

In Towle et al.¹, the anti-cancer activity of two compounds derived from halichondrin B were characterized under in vitro and in vivo conditions. Cytoskeleton’s Custom Services Department participated in this study by screening the compounds for their effectiveness at inhibiting tubulin polymerization in vitro, as determined by IC50 calculations and comparisons against well-characterized tubulin polymerization inhibitors. The in vitro studies contributed to the conclusion that these compounds act as anti-cancer agents through an inhibition of tubulin polymerization¹. The mammalian brain tubulin used in the screens was prepared by Cytoskeleton and used in an absorbance-based polymerization assay modified by Cytoskeleton scientists (Fig. 1A).

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Figure 1. Purity of tubulin and polymerization curves. A. Purity of mammalian brain tubulin and polymerization activity with control buffer, taxol, or vinblastine. B. Purity of HeLa cancer cell tubulin and polymerization activity with control buffer and vinblastine. C. Purity of soybean tubulin and polymerization activity with control buffer, taxol, and trifluralin.

Assay Development and Compound Screening

Similarly, the second citation² involves purification of brain tubulin with or without microtubule-associated proteins (MAPs) along with purification of tubulin from HeLa cancer cells. Cytoskeleton’s Custom Services scientists used these tubulins to evaluate the antimitotic effects of disorazol E1 using modified tubulin polymerization assays developed by Cytoskeleton. Cytoskeleton’s tubulins and polymerization assays (Fig. 1B) were used to calculate IC50 values for this compound, helping to confirm its strong anticancer properties in vitro and qualifying it as a candidate for further in vivo anticancer studies².

Protein Purification and Assay Development

Tresch et al.³ utilized Cytoskeleton’s Custom Services to produce soybean tubulin and develop a modified tubulin polymerization assay. The authors used these reagents to investigate in detail the mode of action of the herbicides flamprop-M-methyl and its biologically active metabolite flamprop, focusing on the herbides’ effects on in vitro plant (soybean) tubulin polymerization. Cytoskeleton, Inc. provided the >90% purified soybean tubulin and polymerization assay kit and protocols (Fig. 1C), significantly contributing to the novel finding that the herbicide flamprop-M-methyl has an antimicrotubule mechanism of action distinct from other herbicides. The classic herbicides work by disrupting microtubule assembly and stability, as demonstrated by in vitro tubulin polymerization assays. In contrast, flamprop-M-methyl does not affect plant tubulin polymerization in vitro; instead, flamprop-M-methyl exerts its herbicidal effects by affecting the organization and orientation of the mitotic spindle and phragmoblast microtubules³.

These citations clearly demonstrate Cytoskeleton’s commitment and ability to partner with a wide-range of scientists to pursue and complete their research projects with timely and unique contributions in the fields of compound screening, protein purification, and assay development. If you are interested in discussing how Cytoskeleton’s Custom Services Department can help your research, send an email to tservice@cytoskeleton.com and let us help you advance your science.

References

M.J. Towle et al. 2001. In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogues of halichondrin B. Cancer Res. 61, 1013-1021.
S. Baasner et al. 2003. Disorazol E1 - A natural compound with outstanding in vitro antitumor activity. American Association for Cancer Meeting. Poster # 2117.
S. Tresch et al. 2008. The herbicide flamprop-M-methyl has a new antimicrotubule mechanism of action. Pest Manag. Sci. 64, 1195-1203.

About Custom Services

Cytoskeleton, Inc. has been a reliable source of compound screening services in the areas of pre-clinical drug development programs and early compound screening in primary HTS projects, as well as secondary screening and compound target validation. We also have extensive experience in gene design and expression with an eye for producing highly purified biological active proteins. Our expertise in protein purification is the basis for the complementary skill of assay design. We have produced many functional assays for kinesins (e.g., Eg5, CenPE, MKLP2), dynein [cytoplasmic]), myosins (e.g., cardiac, smooth, skeletal, and non-muscle isoforms), small G-proteins (e.g., Rho, Arf, Ral families), tubulins (e.g., tumor, plant, and fungal origins), and actin binding proteins; many of them are multi-protein assays that might have protein complexes of 3 or more subunits, e.g., a soluble sarcomere format and the Arp2/3 complex based assay. Our experiences in antibody and ELISA technology complements the cytoskeletal and signal transduction focus. We support all of our services with a dedicated technical services department and years of laboratory experience in the fields of cell biology, cancer biology, and neuroscience.

For more details on these four main areas click on one of the following:

Compound Screening and Drug Development

Protein Purification

Assay Design and Develoment

ELISA and antibody development

If you have an immediate question please send an e-mail describing your requirements to tservice@cytoskeleton.com.

Have questions? Email tservice@cytoskeleton.com or call (303) 322-2254.

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