Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager.

Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both after isoelectric focusing (IEF) and PAGE with a sensitivity higher than or comparable to standard silver staining methods. In addition to the post-labeling methods mentioned above, pre-labeling with the cysteine-specific fluorophore monobromobimane before 2-D PAGE is a sensitive, fast, and cost-effective alternative to existing staining protocols.

A unique charge-coupled device/xenon arc lamp based imaging system for the accurate detection and quantitation of multicolour fluorescence.

In recent years the use of fluorescent dyes in biological applications has dramatically increased. The continual improvement in the capabilities of these fluorescent dyes demands increasingly sensitive detection systems that provide accurate quantitation over a wide linear dynamic range. In the field of proteomics, the detection, quantitation and identification of very low abundance proteins are of extreme importance in understanding cellular processes. Therefore, the instrumentation used to acquire an image of such samples, for spot picking and identification by mass spectrometry, must be sensitive enough to be able, not only, to maximise the sensitivity and dynamic range of the staining dyes but, as importantly, adapt to the ever changing portfolio of fluorescent dyes as they become available. Just as the available fluorescent probes are improving and evolving so are the users application requirements. Therefore, the instrumentation chosen must be flexible to address and adapt to those changing needs. As a result, a highly competitive market for the supply and production of such dyes and the instrumentation for their detection and quantitation have emerged. The instrumentation currently available is based on either laser/photomultiplier tube (PMT) scanning or lamp/charge-coupled device (CCD) based mechanisms. This review briefly discusses the advantages and disadvantages of both System types for fluorescence imaging, gives a technical overview of CCD technology and describes in detail a unique xenon/are lamp CCD based instrument, from PerkinElmer Life Sciences. The Wallac-1442 ARTHUR is unique in its ability to scan both large areas at high resolution and give accurate selectable excitation over the whole of the UV/visible range. It operates by filtering both the excitation and emission wavelengths, providing optimal and accurate measurement and quantitation of virtually any available dye and allows excellent spectral resolution between different fluorophores. This flexibility and excitation accuracy is key to multicolour applications and future adaptation of the instrument to address the application requirements and newly emerging dyes.

A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics.

In order to quantify autoantibodies in the sera of patients with autoimmune disease, we have created a microarray-based immunoassay that allows the simultaneous analysis of 18 known autoantigens. The microarrays contain serial dilutions of the various antigens, thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. The assay is very sensitive and highly specific: as little as 40 fg of a known protein standard can be detected with little or no cross-reactivity to nonspecific proteins. The signal intensities observed from serial dilutions of immobilized antigen correlate well with serial dilutions of autoimmune sera. Miniaturized and highly parallelized immunoassays like these will reduce costs by decreasing reagent consumption and improve efficiency by greatly increasing the number of assays that can be performed with a single serum sample. This system will significantly facilitate and accelerate the diagnostics of autoimmune diseases and can be adapted easily to any other kind of immunoassay.

Patch clamp investigation into the phosphate carrier from Saccharomyces cerevisiae mitochondria.

After heterologous expression in E. coli, functionally active phosphate carrier (PIC) from Saccharomyces cerevisiae mitochondria was purified and reconstituted into giant liposomes and used for patch clamp experiments. Single channel currents across excised patches revealed an anion channel function of the PIC protein. Besides the three transport modes known to date, namely phosphate/phosphate exchange, phosphate/OH exchange and mercurial-induced unidirectional transport, this channel activity represents the fourth transport mode of the PIC. The PIC channel activity was sensitive towards phosphate as its physiological substrate. Phosphate (10 mM) blocked in a specific but reversible manner the PIC channel, suggesting a phosphate-dependent conformational change of the protein into the carrier mode. Furthermore, the current through the channel and its gating activity were affected by divalent cations. In the presence of Ca2+ and Mg2+, the channel displayed a mean conductance of 25 +/- 5 pS whereas 40 +/- 10 pS was observed in the absence of divalent cations. Also, the dwell times in either the open or closed state of the PIC channel appeared to be prolonged in the presence of Ca2+ and Mg2+. The observed PIC channel characteristics are discussed with respect to previously reported electrophysiological in situ measurements on anion channels of the inner mitochondrial membrane. Similarities of the PIC channel to the inner mitochondrial anion channel (IMAC) have been found.

Uptake of creatine phosphate into heart mitochondria: a leak in the creatine shuttle.

CrP uptake into isolated rat heart mitochondria was studied using silicone oil centrifugation. Further, the involvement of the mitochondrial adenine nucleotide translocase was examined by measuring CrP accumulation in mitochondria in the presence of substrates and inhibitors of the ATP/ADP-carrier and by investigating uptake kinetics in liposomes reconstituted with purified bovine heart adenine nucleotide translocase protein. CrP is accumulated in the matrix space of isolated rat heart mitochondria and mitoplasts. The uptake is inhibited by carboxyatractyloside, a specific inhibitor of the mitochondrial adenine nucleotide translocase, and by ADP, phosphoenolpyruvate, 3-phosphoglycerate and pyrophosphate, compounds which are able to bind to the carrier. It is not inhibited when the mitochondrial membrane potential is decreased. CrP is transported into reconstituted liposomes at a rate which is about 3 orders of magnitude lower than the rate for ATP uptake. The transport is sensitive to temperature change and to carboxyatractyloside. It is concluded that CrP is specifically taken up by heart mitochondria via the mitochondrial adenine nucleotide translocase. The transport in mitochondria in situ is facilitated by the close local and functional interaction of the mitochondrial creatine kinase and the adenine nucleotide translocase within contact sites between inner and outer mitochondrial membrane. A certain amount of CrP synthesized by the mitochondrial creatine kinase thus escapes its usage at cytosolic energy consuming processes.

Kinetic and energetic characterization of solute flux through the reconstituted aspartate/glutamate carrier from beef heart mitochondria after modification with mercurials.

The functional switch from specific, coupled antiport to unspecific unidirectional transport (efflux) of the reconstituted aspartate/glutamate carrier from mitochondria after chemical modification with mersalylic acid was investigated in kinetic and energetic terms. The rate of mercurial-induced efflux was determined for a number of solutes which differ from the physiological substrate aspartate in structure, size and charge, namely oxoglutarate, sulfate, glucose, lysine and arginine. These values were compared to the rates of efflux as well as antiport of aspartate. Measurement of the temperature dependence of all rates led to evaluation of the activation energy of the different substrates. The activation energy was similar for all substrates and for both transport modes, whereas the efflux rates could be ordered in the following sequence: anions > uncharged solutes > cations. When extrapolating to Vmax conditions, the resulting turnover numbers for uniport substrates become similar and exceed the turnover numbers for aspartate and glutamate antiport. Trans-inhibition of efflux was only observed in the case of externally added aspartate or glutamate and only for internal anionic substrates (at the cis side), thus indicating that after efflux induction the specificity of the external binding site is fully and that of the internal site is partially retained. The consequence of these results for understanding the transport function of the aspartate/glutamate carrier in the slippage mode (uniport) is discussed in energetic and kinetic terms.