Protein flowing systems

Fung, Y.-S. and Mo, S.-Y., Determination of amino acids and proteins by dualelectrode detection in a flow system, Anal. Chem., 67, 1121, 1995. 

The quantum yield of the mant fluorophore in mant-GDP or mant-GTP increases approximately by 100 % when the molecule changes from the aqueous environment into the nucleotide binding pocket of the Ras protein. Therefore the kinetics of complex formation between nucleotide free Ras and the mant analogues of GDP or GTP could be detected easily in a stopped flow system by an increase in fluorescence signal. 

There are no inherent limitations to the nature of the interaction that can be probed with the FAC method. This too stems from an uncoupling of the binding event and the detector. The method can be applied to simple binary interactions between protein and small molecule, but also to protein-protein interactions, protein-cell interactions and virtually any interaction that can be modeled in a flow system. Some of the more elegant examples include drug interaction with whole cells [12] and membrane-bound receptors from brain homogenates [13]. Ultimately, the limitations are dictated by what can be detected from a stream of column effluent. 

Chance and co-workers have designed a flow system where the protein is continuously pumped optically using a tungsten or xenon flash lamp (764 nm). Using continuous illumination for various times and temperatures. Chance et al. have observed three intermediate states upon MbCO photolysis. At 40 K, a state with a recombination rate constant of 2 x 10 /s has been identified from two slower states with rate constants of 10 /s. 

CFCF, continuous-flow cell-free protein synthesis system PCR, polymerase chain reaction TB, transcription buffer. 

The analysis of libraries by flow cytometry requires special attention to be paid to the protein expression system. An ideal expression system for isolation of live cells by flow cytometry must fulfill the following criteria (Daugherty et al., 1999) ... 

A tubular sonicatlon device was recently reported by Borthwick et al. [93] (see Fig. 3.9). The device requires the addition of no chemical, enzyme or particles that might complicate the subsequent determination step. Furthermore, denaturatlon of target DMA or proteins for detection Is minimized as the device tolerates moderate temperature rises this allows the use of sensitive and specific Immunological detection methods on sonicated biological materials. Because the tubular device Is composed of a piezoelectric resonator made of several material layers, selection of an appropriate operating frequency Is essential to ensure proper performance (i.e. acceptable cell disruption efficiency). This device can be used for batchwise treatment of small sample volumes or In flow systems without the risk of hazardous aerosol formation inherent in probe sonloators. 

Electronic absorption spectroscopy can be used to determine the general structure of porphyins and their derivatives. The oxidation and coordination state of the iron and the identity of the amino acid that ligates the heme can be examined by comparing the absorption spectrum of the protein of interest with the spectra of known heme proteins (45, 65). General characterization with electronic absorption spectroscopy indicated that NO and CO, but not O2, bind to the sGC heme moiety (66). Dynamic studies of ligand binding and dissociation also can be examined with this technique on psec-msec time scales with standard stopped-flow systems. 

Figure 1. Diagram of HPLC-based protein foWing system. Thin lines represent bidirectional computer commimications and data c ture thick lines rraresent fluid/buffer flow towards fraction collector.

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