The PRMM IgG array fabrication with protein G or without protein G and array-based immunoassay procedures.

<p>Six PRMMs were directly labeled with fluorescent dye and probed with antibody slides (aldehyde-derivatized slides coated with or without protein G). Step a: The aldehyde-derivated slide was coated with protein G. Step b: The antibody was printed and immobilized on the protein G-coated slide at 4°C for 2 hours. Step c: The antibody was printed and immobilized on the aldehyde slide at 4°C for 14 hours. Step d: The samples were labeled with fluorescent dye and incubated with the antibody microarray slides at room temperature for 1 hour. Step e: The unbound samples were washed several times. Finally, the signals were directly detected using a fluorescence microarray scanner.</p

Cross-reactivity analysis of the array-based immunoassays with protein G (gray bar) and without protein G (black bar).

<p>Each single PRMM was individually probed with the IgG arrays containing all of the PRMM antibodies: (a) SP, (b) CGRP, (c) NGF, (d) BDNF, (e) TNF-α, and (f) β-endorphin. The error bars represent the standard deviations of four measurements.</p

Sensitivity, dynamic range and LOD for the detection of PRMMs using protein G-facilitated immunoassays.

<p>Note:</p><p>Dynamic range: from LOD to the “saturation point” (beyond which no significant signal increase was observed) of the dose-response curve.</p><p>Sensitivity: (the intensity of saturation point - LOD)/concentration difference.</p

High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System

Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein–protein interactions and enzymatic activity. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases

IgG binding assay of Ru(II)-protein G conjugates.

<p><b>A.</b> Schematic of IgG-binding assays of Ru(II)-protein G conjugates. Normal sheep IgG was immobilized on the 96 well plate, and then Ru(II)-protein G conjugates bound to the Fc region of IgG. <b>B.</b> Effect of molar ratios of SATA to Protein G for conjugation were tested: 10, 15 and 25-fold. Negative control: Ru(II) complex without Protein G.</p

Features of Ru(II) complex.

<p><b>A.</b> The chemical structure of Ru(II) complex, 4-bromophenanthroline bis-2,2′-dipyridine Ruthenium bis (hexafluorophosphate). <b>B.</b> The absorbance and emission spectra of Ru(II) complex. The absorbance spectrum (blue dot-dashed line) was scanned from OD₂₀₀ to OD₆₀₀. The major absorption peak was at 289 nm and minor peak was at 452 nm. Emission spectra were detected from 500 nm to 800 nm and excited at 452 nm (black solid line) and 289 nm (red dashed line), respectively. The Ru(II) complex using 289 nm excitation wavelength showed weak fluorescent signal. On the other hand, the Ru(II) complex using 452 nm excitation wavelength showed large fluorescent intensity and the emission peak wavelength of Ru(II) complex was at 602 nm. <b>C.</b> Fluorescence decay curve of Ru(II) complex.</p

SDS-PAGE of protein G and Ru(II)-protein G conjugates.

<p>The Ru(II) complex was successfully conjugated to the SATA modified protein G and higher molar ratio of SATA to protein G provided higher conjugation efficiency. SATA (+) represents 25-fold molar ratio to protein G. SATA (++) represents 50-fold molar ratio to protein G. Negative control: conjugation without protein G (lane F and G). SATA-Ru(II) complex was expected to form in the negative control.</p

Detection of purified recombinant proteins by Ru(II)-protein G conjugates.

<p><b>A.</b> Schematic of Ru(II)-protein G conjugates for detecting histidine-tagged protein. The purified histidine-tagged protein BasR was first immobilized on the 96 well plate and then recognized by anti-His antibody. Finally, the Ru(II)-protein G conjugates bound to the Fc region of anti-His antibody. <b>B.</b> Comparison of Ru(II)-protein G conjugates and Ru(II) complex for detecting histidine-tagged protein BasR. Ru(II)-protein G conjugates showed approximate 8-fold fluorescent signal compared with Ru(II) complexes (negative control) in the assay. <b>C.</b> Dose response of the histidine-tagged recombinant protein. The linear dynamic range of was from 0 to 10 µg/ml (R² = 0.96).</p

Schematics of conjugation between Ru(II) complex and protein G.

<p>The succinimide group of SATA reacted to primary amines of protein G and forms SATA modified protein G. Then, the SATA modified protein G was deacetylated by hydroxylamine. The resulting sulfhydryl modified protein G was conjugated with Ru(II) complex to form Ru(II)-protein G conjugates.</p

Finger Probe Array for Topography-Tolerant Scanning Electrochemical Microscopy of Extended Samples

Scanning electrochemical microscopy with soft microelectrode array probes has recently been used to enable reactivity imaging of extended areas and to compensate sample corrugation perpendicular to the scanning direction. Here, the use of a new type of microelectrode arrays is described in which each individual microelectrode can independently compensate corrugations of the sample surface. It consists of conventional Pt microelectrodes enclosed in an insulating glass sheath. The microelectrodes are individually fixed to a new holder system by magnetic forces. The concept was tested using a large 3D sample with heights up to 12 μm specially prepared by inkjet printing. The microelectrodes follow the topography in a constant working distance independently from each other while exerting low pressure on the surface