HTS & Bulk

CytoDYNAMIX Screens™ -
High throughput assays as a service or product

CytoDYNAMIX Screens™ are extremely robust high throughput assays for use in drug discovery. Cytoskeleton, Inc. offers a variety of high throughput assays that can also be used on a small scale for verification and general research purposes.

Most of our HTS assays are available both as products and as custom services that we can provide. Please contact our Technical Service group (tservice@cytoskeleton.com) for more detailed information about your options.

There is a continuous improvement in existing HTS assays and development of novel assays at Cytoskeleton, Inc.  If you have an interest in one of these assays or you need an assay that you do not see here, please contact our Technical Service group (tservice@cytoskeleton.com).

HTS assays for ATPases, GTPases or phosphate measurements
HTS assays for actin and actin binding proteins
HTS assays for motor proteins
HTS assays for small GTPases
HTS assays for tubulin and tubulin-associated proteins

HTS assays for ATPases, GTPases or phosphate measurements
Many processes inside the cell are dependent on nucleotide hydrolysis, which results in inorganic phosphate (P_i) liberation. Measurement of P_i generation by ATPases and GTPases is a simple, cost-effective, functional assay for this class of important enzymes.  Cytoskeleton, Inc. provides several nucleoside triphosphatase (NTPase) assays for HTS applications. There is a choice of a non-radioactive endpoint assay with a 1 µM phosphate limit of detection (Cat. # BK054), which is good for moderate or high activity NTPases (K_cat >0.05). There is a radioactive filter-based endpoint assay (Cat. # BK055) with a 10 nM detection limit that is good for small G-proteins and microtubule polymerizations which have low GTPase activity (K_cat <0.01). Please see the About phosphate assays page for more details on our phosphate assays.

ATPase Kinetic ELIPA™ Assay Kit (cat# BK051)
CytoPhos™ Endpoint Phosphate Assay (cat# BK054)
EasyRad™ Endpoint Phosphate Assay (cat# BK055)
GTPase Kinetic ELIPA™ Assay Kit (cat# BK052)
HTS kinesin ATPase Endpoint Assay Biochem Kit (cat# BK053)

Kinesin ELIPA Biochem Kit (cat# BK060)

RhoGAP Assay Biochem Kit (cat# BK105)

HTS assays for actin and actin binding proteins
The actin CytoDYNAMIX Screen™ series are high throughput drug development screens utilizing actin proteins, isotypes of actin and actin binding proteins such as WASP, cofilin and profilin. These screens are helping to develop the next generation of drugs targeting cancer, neurological disorders, inflammation and infection. These screens are based on proprietary techniques developed at Cytoskeleton, Inc. They are only available as a custom screen service. Future screens will be introduced utilizing the molecular motors of microfilaments. If you would like more information about these screens, please contact our Technical Service group (tservice@cytoskeleton.com).

Available screens include:
CytoDYNAMIX Screen™ 20: Skeletal or Cardiac Muscle Actin Polymerization Assay
CytoDYNAMIX Screen™ 21: Non-muscle Actin Polymerization Assay
CytoDYNAMIX Screen™ 22: Plant Actin Polymerization Assay
CytoDYNAMIX Screen™ 23: Actin Isotype Polymerization Assay
CytoDYNAMIX Screen™ 24: Actin Binding ELISA-type Assay
CytoDYNAMIX Screen™ 25: Actin Binding Filter Based Assay
CytoDYNAMIX Screen™ 26: Actin Binding Protein Assay with Actin Polymerization (cofilin, profilin, WASP, etc.)

HTS assays for motor proteins
Kinesins are a group of related molecular motors that utilize the energy of ATP hydrolysis to transport cargo such as chromosomes and vesicles along microtubule tracks. Functionally kinesins can be divided into two major groups, those involved in vesicle transport and membrane organization and those involved in mitosis. The central role of kinesins in cell division makes them of great interest as anti-mitotic targets. Of critical importance to the premise of kinesins as drug targets is the fact that all available data indicate that antibodies and small molecules directed against mitotic kinesins specifically inhibit the mitotic process but have no effect upon kinesin vesicle transport functions. The fact that kinesins represent a new anti-mitotic target and that they are expressed predominantly during cell division suggests that they may be superior to and/or complementary to the current highly popular anti-tubulin drugs such as paclitaxel.  To meet the growing demand for purified motor proteins and associated reagents, Cytoskeleton introduced Cytoskeleton Motor Werks™, a comprehensive line of purified motor proteins, antibodies and assays that aid motor protein research.

For HTS applications, Cytoskeleton offers a simple, colorimetric ATPase assay (Cat. # BK053). The assay measures microtubule (MT)-activated kinesin motor ATPase activity. The simplicity of the assay lies in the fact that it uses a single step malachite-based detection system and pre-formed MTs that require no preparation prior to the assay (Cat. # MT002). The rigorously quality-controlled MTs also provide high assay reproducibility.  Kinesin motors are available separately in milligram (mg) quantities and pre-formed MTs are also available separately in 2 mg, 10 mg and bulk sizes. In addition to mitotic motor proteins, several non-mitotic (vesicle transport) motors are available. These are suitable as controls in secondary screens such as the kinesin ELIPA Biochem Kit (Cat. # BK060). The panel of kinesins available are representative of the major classes of kinesin proteins and therefore offer an excellent screening panel for target specificity.

Motor proteins are members of the large family of ATP binding proteins, which also includes kinases and DNA polymerases among others. Well characterized ATP analog libraries can therefore be used as a starting point for discovering inhibitors of the kinesin ATPase proteins. Such a familial approach to drug discovery promises to be efficient and highly cost-effective, yielding high quality hits. More information can be found in this review of the use of ATP analog libraries (Chene, 2003. Expert Opin. Ther. Targets. 7, 453-461).

Kinesin ELIPA™ Biochem Kit (BK060)
Kinesin HTS ATPase Endpoint Assay (BK053)

HTS assays for small G-proteins
The activity and regulation of small G-proteins has been shown to play an important role in the development and progression of a number of diseases such as cancer, cardiovascular disorders, inflammation and neurological disorders. Targeting G-proteins, their regulators and their downstream effectors is therefore becoming an important area of drug discovery. See the main G-protein page for general information about these proteins. Cytoskeleton, Inc. provides HTS format assays to measure  in vitro GEF, GAP and G-protein-effector interactions for drug discovery.  In addition, Cytoskeleton, Inc. offers a large number of purified and tagged wild-type, dominant-negative and constitutively-active small G-proteins.

RhoGAP Assay Biochem Kit (cat# BK105)
RhoGEF Exchange Assay Biochem Kit (cat# BK100)

HTS assays for tubulin and tubulin-associated proteins
With microtubules playing a central and vital role in cell division as well as vesicle and cargo transport in cells, targeting microtubule dynamics is an obvious and well-established area of drug discovery. Cytoskeleton, Inc. is the world leader in providing the scientific community with purified tubulin proteins and tubulin-related assays.

The absorbance-based tubulin polymerization assay (cat# BK004P) is well-established in the pharmaceutical sector and is highly referenced in the scientific literature. It is supported by patented technology for lyophilized tubulin and each customer obtains a free license to use these preparations for screening and drug development purposes. A second absorbance-based tubulin polymerizaton assay (cat# BK006P) uses highly purified tubulin, which is a good secondary screen for determining IC50s and tubulin specificity. The fluorescence-based tubulin polymerization assay (cat. # BK011P) is less established in the field but is a good alternative for HTS because of the price savings over the absorbance based assay on a price per reaction basis. 

For more selective anti-cancer drug development, a very exciting secondary screen can be achieved using purified tubulins from various cancer cell lines. Currently, cancer tubulin from HeLa and MCF-7 cell lines can be used in the fluorescence polymerization assay (cat# H001 and H005, respectively). It has been shown that anti-cancer drugs have different affinities for these tubulins compared to bovine brain (neuronal) tubulin. This indicates that a drug development program aimed at increasing the affinity for cancer cell line tubulin over neuronal tubulin may lead to greater targeting efficiency toward cancer cells.

We also provide HTS assays for other types of tubulins, such as fungal tubulins, plant tubulins (cat# BK010S) or bacterial tubulin analogs (cat# FTZ01 and others soon to come).  If you are interested in assaying a specific tubulin not listed on our website, please contact our Technical Service group (tservice@cytoskeleton.com) to discuss how we can provide you with the specific tubulin type you want.

Tubulin Polymerization HTS Assay using >97% pure tubulin, OD-based, porcine (BK004P)
Tubulin Polymerization HTS Assay using >99% pure tubulin, fluorescence-based, porcine (BK011P)
Tubulin Polymerization HTS Assay using >99% pure tubulin, OD-based, porcine (BK006P)

Tubulin Ligand Competition Assay (CDS15P)

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Question 1:  What kind of references do you have for Cytoskeleton's products used in drug screening?

Question 2:   What kind of discounts can I expect for bulk purchases? 

Question 3:  Will Cytoskeleton help me develop my drug screen?

Question 1: What kind of references do you have for Cytoskeleton's products used in drug screening? 

Answer 1:Cytoskeleton, Inc. has been a reliable source of protein reagents for drug screening for nearly two decades. Past clients include:

Merck         Eli Lilly         Amgen         Abbott Labs        GlaxoSmithKline

Genentech       Johnson & Johnson       Bristol -Meyers Squibb        Pfizer        Wyeth

 We support all of our products with a dedicated technical services department and years of experience in the fields of cell biology, cancer biology and neuroscience.

Some of the bulk purchases intended for screening projects are related to proprietary research, potentially limiting the number of citations available.  Here are a select few recent publications that used our motor protein and/or tubulin products.

Mi et al., 2008.  Covalent Binding to Tubulin by Isothiocyanates: A mechanism of cell growth arrest and apoptosis.  J. Biol. Chem. 283, 22136–22146.

Gorenstein et al., 2010. Reducing the multidimensionality of high-content screening into versatile powerful descriptors. BioTechniques. 49, 663-665.

Meng et al., 2008.  A novel class of tubulin inhibitors that exhibit potent antiproliferation and in vitro vessel-disrupting activity. Cancer Chemother. Pharmacol. 61, 953-963.

Motor protein antibody)  Endo et al., 2009. Truncated form of tenascin-X, XB-S, interacts with mitotic motor kinesin Eg5. Mol. Cell. Biochem. 320, 53-66.

Xing et al., 2011.  A potent chemotherapeutic strategy in prostate cancer: S-(methoxytrityl)-L-cysteine, a novel Eg5 inhibitor. Asian J. Androl. 13, 236-241.

Rodriguez et al., 2011.  Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity. Bioorg. Med Chem. 19, 5446-5453.

Grigoryan  et al., 2009.  Covalent binding of the organophosphorus agent FP-biotin to tyrosine in eight proteins that have no active site serine. Chem. Biol. Interact. 180, 492–498.

Dinu et al. 2009.  Tubulin Encapsulation of Carbon Nanotubes into Functional Hybrid Assemblies. Small. 5, 310–315.

Wasylycia et al., 2008.  Nano-biopower supplies for biomolecular motors: the use of metabolic pathway-based fuel generating systems in microfluidic devices.  Lab Chip. 8, 979-982.

Question 2: What kind of discounts can I expect for bulk purchases?  

Answer 2: Cytoskeleton, Inc. wants to help your project be successful in any way that we can.  We are aware of budget constraints on projects and will do our best to work with you to reach a pricing structure amenable to everyone.  The actual discount will depend on the particular product, but 20-50% discounts are not uncommon for quantities of 50 or more.

Question 3: Will Cytoskeleton help me develop my drug screen?

Answer 3: Cytoskeleton, Inc. will help with optimizing conditions prior to initiating the experimental assays.  We can offer suggestions and guidance for (i) choosing optimal experimental conditions, (ii) designating optimal equipment settings and (iii) performing equipment trouble-shooting.