Substrate labeling

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

The hydrolysis of a nonfluorescent enzyme substrate to a fluorescent product is widely utilized to measure the activity of a large number of enzymes. Binding of enzyme substrates by antibodies often protects the enzymatically labile bond from hydrolysis. By the combination of these two formats, the substrate-labeled fluorescent immunoassay (SLFIA) was developed. ... 

Figure 6.2. Chemical structure of 8-[3-(7-/)-galactosylcoumarin-3-carboxyamido )propyl ] theophylline (B) used in the homogeneous substrate-labeled fluorescent immunoassay for theophylline (A). (Reprinted from Ref. 3, with permission from the American Association for Clinical Chemistry.)...

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

The apparent retinal influx clearance,. Kin,retina expressed as mL/(min g retina), of the test substrate labeled with either [3 H] or [14C] from the circulating blood to the retina is determined by integration plot analysis. In brief, rats are anesthetized, followed by injection of the test substrate (e.g., an [3H]-labeled compound, about 10 /u.Ci/head) into the femoral vein. After collection of plasma samples, rats are decapitated and the retinas removed. The retinas are dissolved in 2 N NaOH and subsequently neutralized with 2 N HC1. The radioactivity of retinal cell lysates is measured by liquid scintillation spectrometry. As an index of the retinal distribution characteristics of the radiolabeled test substrate, the apparent retina-to-plasma concentration ratio (Vd) as a function of time is used. This ratio [Vd(Q] (mL/g retina) is defined as the amount of [3H] per gram retina divided by that per milliliter plasma, calculated over the time-period of the experiment. The Kjn,retina can be described by the following relationship ... 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Islam K, Zheng W, Yu H et al (2011) Expanding cofactor repertoire of protein lysine methyltransferase for substrate labeling. ACS Chem Biol 6(7) 679-684... 

Fig. 4. Schematic representation of the principle of substrate-labeled fluorescent immunoassay (tda, Ames). [Cited and modified from Fig. B-5, Miyai, K., in Miyai etal., eds. (M10).]...

An artificial substrate labeled with a strong UV-absorbing chromophore was synthesized. This substrate and its product, obtained by hydrolysis of the ADP-ribosyl moiety, were separated on a Zorbax-ODS reversed-phase CI8 column (4.6 mm X 250 mm). The initial mobile phase was 70% 0.0435 M sodium acetate (pH 4.0) and 30% acetonitrile. The percentage of acetonitrile... 

The application of substrates labeled with various chromophores has become more and more popular in recent years. It has been already shown that proteolytic activity assays using dyed proteins have a long tradition (see 6.1). Their enforcement has not, however, been simple. There have been objections (often correct) against a discutable correlation between the colour intensity and the number of split peptide bonds. This disadvantage can be overcome by specific binding of a certain dye to the amino acid involved in the proteolytic breakdown of the protease being assayed. 

Therefore, due to the practical limitations described above, the use of a non-fluo-rescent acceptor (usually called a quencher) is still the most popular way to use FRET in HTS applications. Most of the time, FRET is used to probe a protease activity using a peptide substrate labelled at each end of its sequence with the fluorescent donor and the quencher. In that case, the FRET readout is an increase in the donor fluorescence emission upon peptide cleavage by the protease. " However, as discussed above, such FRET readouts can lead to a significant number of false positive results in HTS. 

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