Generating addressable protein microarrays with PROfusion™ covalent mRNA-protein fusion technology

Weng, Shawn; Gu, Ke; Hammond, Philip W.; Lohse, Peter; Rise, Cecil; Wagner, Richard W.; Wright, Martin C.; Kuimelis, Robert G.

doi:10.1002/1615-9861(200201)2:1<48::aid-prot48>3.0.co;2-i

Cited by 96 publications

(36 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is conventionally called ORFeome cloning Vaglio et al 2003;Brasch et al 2004). If such a set of cDNAs becomes available, it would be straightforward to prepare probe microbeads, e.g., by self-assembling methods (He and Taussig 2001;Weng et al 2002;Ramachandran et al 2004). In the same way, the preparation of fluorescently labeled target proteins also becomes straightforward as demonstrated in this study (Figs.…”

Section: Discussionmentioning

confidence: 99%

Development of a microscopic platform for real-time monitoring of biomolecular interactions

Sasuga

Tani²,

Hayashi³

et al. 2005

Genome Res.

View full text Add to dashboard Cite

We developed a new microscopic platform for the real-time analysis of molecular interactions by combining microbead-tagging techniques with total internal reflection fluorescent microscopy (TIRFM). The optical manipulation of probe microbeads, followed by photo immobilization on a solid surface, enabled us to generate arrays with extremely high density (>100 microbeads in a 25 µm × 25 µm area), and TIRFM made it possible to monitor the binding reactions of fluorescently labeled targets onto probe microbeads without removal of free targets. We demonstrated the high performance of this platform through analyses of interactions between antigen and antibody and between small compounds and proteins. Then, recombinant protein levels in total cellular lysates of Escherichia coli were quantified from the association kinetics using antibody-immobilized microbead arrays, which served as a model for a protein-profiling array. Furthermore, in combination with in vitro synthesis-coupled protein labeling, we could kinematically analyze the interaction of nuclear factor B (p50) with DNA. These results demonstrated that this platform enabled us to: (1) monitor binding processes of fluorescently labeled targets to multiple probes in real-time without removal of free targets, (2) determine concentrations of free targets only from the association kinetics at an early phase, and (3) greatly reduce the required volume of the target solution, in principle to subnanoliter, for molecular interaction analysis. The unique features of this microbead-based microarray system open the way to explore molecular interactions with a wide range of affinities in extremely small volumes of target solutions, such as extracts from single cells.

show abstract

Section: Discussionmentioning

confidence: 99%

Development of a microscopic platform for real-time monitoring of biomolecular interactions

Sasuga

Tani²,

Hayashi³

et al. 2005

Genome Res.

View full text Add to dashboard Cite

show abstract

“…There exists a number of signal amplification techniques, compatible with protein arrays. These include more traditional ELISA (enzyme-linked immunosorbent assay) or ECL (enhanced chemiluminescence)-based approaches, a Rolling Circle Amplification (RCAT), capable of microchip format applications and up to 4 orders of magniAssay Formats for High-Throughput Affinity Arrays tude signal amplification 16 , or the use of protein-DNA fusions [17][18] , where proteins can be labelled to a much higher degree through the associated DNAs thus enabling detection of protein molecules present in low abundance on an array. To maintain quantitative character of an assay, a more efficient protein labelling (i.e., through protein fusions, etc.)…”

Section: Discussionmentioning

confidence: 99%

Competitive Assay Formats for High-Throughput Affinity Arrays

Barry

Diggle²,

Terrett³

et al. 2003

SLAS Discovery

View full text Add to dashboard Cite

The authors describe a novel method for the quantitation of differential levels of biomolecules using unlabeled samples and protein-binding arrays for assessing differential expression. Traditional affinity arrays, whether in microplates or protein microarrays, suffer from a few common problems-a shortage of characterized antibodies and highly variable affinities for those available. Also, the assayed proteins could be present in a wide range of concentrations and physicochemical properties, so that it becomes an onerous task to optimize assay conditions for each antibody-antigen pair. Currently, this restricts parallel affinity assays to a low number of carefully selected antibodies and restricts the development of highly multiplexed parallel affinity assays. A displacement strategy allows the use of a much wider range of antibodies, reducing the requirement for matched affinities. The competitive assays described here also show a much higher tolerance for nonspecific background noise. The range of assayed protein concentrations is only limited by the sensitivity of the detection system used. (Journal of Biomolecular Screening 2003:257-263)

show abstract

“…The fusion of RNA to proteins and the following hybridization to the respective complementary oligonucleotide target on a linker modified slide surface of type 2B was described by Weng et al [99]. This technique positions the RNA-protein fusion in a uniform orientation thereby avoiding the delicate contact of the proteins with the slide surface.…”

Section: Immobilization Strategies For Different Probesmentioning

confidence: 99%

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

Sobek¹,

Bartscherer²,

Jacob³

et al. 2006

CCHTS

110

View full text Add to dashboard Cite

Over the last years microarray technology has become one of the principal platform technologies for the highthroughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.

show abstract

Generating addressable protein microarrays with PROfusion™ covalent mRNA-protein fusion technology

Cited by 96 publications

References 32 publications

Development of a microscopic platform for real-time monitoring of biomolecular interactions

Development of a microscopic platform for real-time monitoring of biomolecular interactions

Competitive Assay Formats for High-Throughput Affinity Arrays

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

Contact Info

Product

Resources

About