High throughput assays as a service or product
CytoDYNAMIX Screens™ are highly robust high throughput assays for use in drug discovery. Cytoskeleton, Inc. offers a variety of high through-put 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 services that we can provide. Please contact our technical support for more detailed information about your options.
There is a continual improvement and novel design process of HTS 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 support.
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 (Pi) liberation. Measurement of Pi generation by ATPases & GTPases is a simple, cost effective functional assay for this class of important enzymes. Cytoskeleton, Inc. provides several NTPase assays for HTS applications. There is a choice of non-radioactive endpoint assay with a 1 µM phosphate limit of detection (BK054), which is good for moderate or high activity NTPases (Kcat >0.05). There is a radioactive filter based endpoint assay (BK055) with 10 nM detection limit that is good for small G-proteins and microtubule polymerizations, which have low GTPase activity (Kcat <0.01). See the About phosphate assays page for more details on our phosphate assays
ATPase Kinetic ELIPA™ Assay Kit (BK051)
CytoPhos™ Endpoint Phosphate Assay (BK054)
EasyRad™ Endpoint Phosphate Assay (BK055)
GTPase Kinetic ELIPA™ Assay Kit (BK052)
HTS kinesin ATPase Endpoint Assay Biochem Kit (BK053)
HTS assays for actin and actin binding proteins
The actin CytoDYNAMIX Screen™ series are high throughput drug development screens based on 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 targeted to cancer, neurological disorders, inflammation and infection. These screens are based on proprietary techniques developed at Cytoskeleton, Inc. They are only available as a screen service. Future screens will be introduced based on molecular motors of microfilaments. If you would like more information about these screens, please contact our technical support
Available services 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 targets for anti-mitotics. Of critical importance to the premise of kinesins as drug targets is the fact that all available data indicates 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.
The Cytoskeleton Motor Werks™ range of products provides a comprehensive line of 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. # MT001). The rigorously quality controlled MTs also account for high assay reproducibility. Kinesin motors are available separately in mg quantities and pre-formed MTs are also available separately in 2 mg, 10 mg and bulk sizes. In addition to mitotic motor proteins we provide several non-mitotic (vesicle transport) motors available. These are suitable as controls in secondary screens such as 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 in 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 a recent review of the use of ATP analog libraries (Chene, P. Expert Opin. Ther. Targets (2003) 7: 453-461).
Kinesin ELIPA™ kit (BK060)
Kinesin HTS ATPase end point 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 vivo activities of small G-proteins as well as in vitro drug discovery HTS assays for GEFs, GAPs and G-protein - effector interactions.
G-LISA™ RhoA activation assay, absorbance based (BK124)
G-LISA™ RhoA activation assay, luminometry based (BK121)
G-LISA™ RhoA/B/C activation assay, absorbance based (BK123)
Rho - effector in vitro interaction HTS G-LISA™ (BK122)
RhoGAP assay (BK105)
RhoGEF exchange assay (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. are the world leaders at providing the scientific community with tubulin proteins and tubulin related assays.
The optical density based tubulin polymerization assay (Cat. # CDS01) is well established in the pharmaceutical sector and is highly referenced in 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. The CDS03 tubulin polymerization assay uses highly purified tubulin, which is a good secondary screen for determining IC50s and tubulin specificity. The fluorescence tubulin polymerization assay (Cat. # BK011) is less established in the field but is a good alternative for HTS because of the price savings over the absorbance based assay.
For more selective anti-cancer drug development a very exciting secondary screen can be achieved using Cancer Cell Line Tubulin. Currently, HeLa and MCF-7 cell line tubulins can be used in the fluorescence polymerization assay (Cat. # H001-B 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 (e.g. BK010) or bacterial tubulin analogs. If you are interested in assaying a specific tubulin not listed on our website, please contact our technical support to discuss how we can provide you with the specific tubulin type you want.
A more defined mechanism of action can be obtained by using the microtubule depolymerization assay (Cat. # BK012), which measures specific interaction with microtubules (compared to tubulin monomer) and creates a Vmax for depolymerization of microtubules.
Tubulin polymerization HTS assay using >97% pure tubulin, OD based (CDS01)
Tubulin polymerization HTS assay using >99% pure tubulin, fluorescence based (BK011)
Tubulin polymerization HTS assay using >99% pure tubulin, OD based (CDS03)
