Assays volume

Assay volumes usually range from 3 pL (for 1536-well MTPs) to 50 pL (384-well MTPs). Within a given total assay volume, smaller volumes of reagents are added. Frequently, we find it convenient to add reagents into the assay in equivalent volumes of assay buffer. As an example, for a 15-pL assay, one might add 5 pL of compound solution, 5 pL of enzyme stock solution, 5 pL of substrate mix, followed by 10 pL of quench solution in a stop buffer. For kinase assays, the stop buffer may be EDTA and for phosphatase assays, sodium orthovanadate. 

Assay miniaturization helps to reduce the consumption of very expensive assay reagents. The problems encountered as one attempts to miniaturize an assay relate to the change in surface-to-vol-ume ratio, lowered sensitivity, and low volume dispensing of materials. For instance, as one moves to smaller volumes, the surfaces available for unspecific binding increase relative to the volume. Furthermore, the smaller the volume, the less the amount of product-sensing material can be added thus the sensitivity of the assay is reduced. 

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HTS is usually carried out it multiwell plates and the industry has settled on certain standard formats. One key standard is that most screening assays are carried out in 96 (8 12) or 384 (16 24) well plates with a standard footprint. This standard is important because most automation is optimized for use with these plates and attendant assay volumes in the 100 pL range. Furthermore, source plates containing test compound are typically stored in a similar configuration, allowing for more efficient transfer of test compound from the source plate to the assay plate. Various types of plates are available to match different assay formats, including... 

Standard curves performed under our defined radioimmunoassay conditions ([ H]PbTx-3 = 1 nM, antiserum dilution = 1 2000, assay volume = 1 ml) demonstrated the ability of this antiserum to bind equally to PbTx-2 and PbTx-3, suggesting specificity for the cyclic polyether backbone region of the molecule (Figure 8). The linear portion of the curve indicated a lower detection limit of 0.2-0.5 ng in saline buffer under these conditions. Evaluation of this assay for use with biological fluids and tissue extracts is underway. 

Membranes (50 pi in a total assay volume of 100 pi) were incubated with UDP-Gal (0.1 mM) and MgSO (10 mM) in 25 mM Tris-HCl buffer pH 7.5, for 10 or 60 min. Reactions were stopped by heating at 100°C for 3 min. Lupin galactan (0.1 mg) was added as a 0.1% solution, methanol was added to give a final concentration of 70% by volume, and the tubes were capped, heated at 70°C for 5 min and centrifuged (13000g 5 min). Supernatants were discarded or retained for analysis. Pellets were washed twice more with 70% methanol at 70 C and the supernatants were discarded. The final pellets were either dissolved in preparation for scintillation counting, or were suspended in water and freeze dried in preparation for analysis. 

In order for the indicator reaction to parallel the test reaction the ratio, Vmax/ATm, for the indicator enzyme should be significantly greater than the same ratio for the test enzyme. The value for Vmax is the only variable in the equation and is determined by the amount of enzyme present, usually quoted in units per assay volume. An increase by a factor of 100 is usually recommended for each stage and will permit concentrations of approximately 1/100 of the Km value to give a rate comparable to the test reaction rate. It may be necessary, if pH or inhibition effects reduce the activity of the enzyme by a known proportion, to increase further the amount of enzyme added to the assay. Table 8.3 lists some examples of coupled and direct assays for a range of enzymes. 

Knowing the total assay volume the actual amount of product can be calculated from the concentration alue. gi ing the value for the numlicr of units of enzyme activity in the assay. 

If 1.0 ml of enzyme preparation in a total assay volume of 5.0 ml results in the formation of a concentration of 0.3 mmol l-1 of product in 10 minutes, what is the activity of the enzyme preparation in katal ml 1 ... 

An often-noted paradox in HTS is that as microplate wells and assay volumes have become smaller, automated systems to accommodate them have grown ever larger. This truth raises an important point Because HTS is not a bottleneck in the drug-discovery... 

Interference by other thiols (e.g. as enzyme buffer components) Homogeneous assay, commerically available components Heterogeneous assay, very low assay volume possible Heterogeneous assay... 

Heterogeneous assay, very low assay volume possible Coupled enzymatic assay... 

Coal Rank Gray-King Assay Volume of gas... 

Coal Conditions of Treatment Gray-Kina Assay Volume of... 

Biological test in which the severity of the toxic effect caused by a test material is measured by the response of living organisms. Synonyms biotest, toxicity test, toxicity assay. Volume 2(8). 

Determined on the basis of loss of cell membrane permeability in response to a deliberate modification in culture conditions. Cell viability can be determined by either neutral red or fluorescein diacetate retention assays. Volume 1(14,15). 

Fig. 1. Assay for progestin receptors in human breast cancer cytosol. A. Saturation analysis. Total ( — ) and aspecific (o—o) binding are measured Specific binding (solid line) is calculated by subtraction. B. Scatchard plot derived from the binding data by the method of Chamness and McGuire [8]. Numerical data protein concentration of cytosol. 1.8 mg/ml assay volume, 100 /d cytosol volume, 50 /ul. Calculated values Kd = 0.76 nmol/l progestin receptor content, 510 fmol/mg protein.

Microassays were used in which the final assay volume was 10 pL. Microvolumes were dispensed by using a 100 pL Hamilton syringe and a repeating dispenser. Reactions were carried out in 96-well polystyrene microplates. A typical well contained 2 pL of sodium phosphate buffer, 2 pL of water, and... 

An AlphaScreen assay is generally conducted in a 384-well plate in a total volume of 25 ]xL, but can be scaled down to the 1536-well format with assay volumes of 5 pL. Since the donor beads are light sensitive, the assay has to be performed under dimmed light After all components have been added, the luminescence signal is measured, usually after an incubation of 1 h. 

A precondition for miniatiuization of the assay volume to a few microliter is the availability of reliable and versatile liquid-handling tools for precise metering and delivery of microliter and nanoliter aliquots of compound solutions, assay reagents, and biological materials. Low-volume liquid handlers in the life sciences utilize a variety of basic microfluidic methods for precise dosage of the aliquot volume being transferred, including ... 

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