Fluorescence labels in kinases

Fluorescence Labels in Kinases A High-Throughput Kinase Binding Assay for the Identification of DFG-Out Binding Ligands... 

Since the Schiff base formation is reversible, it should be reduced by sodium borohydride for the fixation of the label. The rate of the reduction of the Schiff base becomes slow as the number of the phosphate groups of the label increases. However, except for adenylate kinase, the NP -PL bound to the proteins were easily fixed by borohydride reduction. After reductive fixation, labeled proteins are cleaved by appropriate methods. The labeled lysine is cleaved by neither trypsin nor lysyl endopeptidase. There are at least three ways to detect the labeled peptide during isolation 1) use of radioactive reagent, 2) use of radioactive sodium borohydride for reduction of the Schiff base, and 3) use of fluorescence derived from the pyridoxyl moiety of the reagent (excitation at 295 nm and emission at 390 nm at acidic pH). The labeled lysyl residue is not positively identified in the amino acid sequence analysis. However, the presence of the label in the peptide isolated can be confirmed by the presence of pyridoxyl lysine in the amino acid analysis. 

The newly available Odyssey and Aerius infrared imaging systems make it possible to probe whole cells with two-color infrared fluorescently labeled antibodies (anti-phosphopeptide, for example) that are used to detect changes in intracellular kinase signaling (Chen et al., 2005). The advantage of using infrared-labeled probes lies in the increased sensitivity and dynamic range and consequent reduction in the use of reagents. 

Fig. 9. Array-based autophosphorylation assays. Here an array of 80 human protein kinases was printed in duplicate and assayed for autophosphorylation activity, (a) Kinase buffer only, (b) Kinase buffer containing 100 pJVI ATP. The assays were developed using a fluorescently labelled anti-phosphotyrosine antibody and revealed ATP-dependent, on-array...

More recently, a number of reports have appeared demonstrating that phosphorylation could be measured in a microarray format using a known kinase-substrate pair. Two different detection approaches have generally been used, the first makes use of [y- / P]-ATP to label the immobilized substrate with a radioactive phosphate, which can be detected by autoradiography, phosphor imager, or silver staining [9,13,75-79]. The second detection method relies on phosphospecific antibodies, which are fluorescently labeled [13,51,58,76,80]. While the fluorescent detection may be preferable as it avoids working with radioactive ATP, it has been shown that only monoclonal anti-phosphotyrosine antibodies showed reliable results [76]. Alternatively, fluorescently labeled phosphor-chelators have been used to detect the phosphorylated peptide in an array [81]. Preliminary results have also been reported for mass spectrometry detection and surface plasmon detection (vide infra). 

Another popular assay format for kinase assays is the Lanthascreen. This format is a variation on the LANCE assay, but employs Tb as the cryptate. In this format N-terminally fluorescently tagged peptide substrate (acceptor) is phosphorylated by the kinase. Next, a phophospecific antibody which is labeled with terbium binds specifically to the phosphorylated product, placing the donor and acceptor in close proximity, generating a signal [25]. 

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

Zeiss have developed a high-throughput reader system (the Plate Vision multimode reader) for microwell plates that is capable of fast time-resolved fluorescence (Fast-TRF) imaging with nanosecond resolution [ 189] (Fig. 28), which has found applications in pharmaceutical screening. This microplate imager is suitable for time-resolved RET assays. Kinase assays, for instance, can be performed with antibodies labeled with a Ru(batho)2bipy complex or... 

While protein kinases are responsible for the phosphorylation of their substrates, protein phosphatases perform the opposite duty, removing phosphate groups from their substrates, thus countering the functional impact of the kinases. The two major types of protein phosphatases are the serine/threonine phosphatases and tyrosine phosphatases. Several natural compounds with potent serine/threonine phosphatase inhibitory activity have been identified, including the cyanobacterial metabolite microcystin [105,106]. This compound labels its targets via a Michael addition of a noncatalytic active site cysteine residue with an acceptor in the macrocyclic peptide backbone [107]. A fluorescent probe based on microcystin was synthesized by Shreder et al., and its use in Jurkat lysates identified two previously undescribed phosphatase targets of microcystin, PP-4 and PP-5 [108]. Whereas serine/threonine... 

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