Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protein Array Analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2002

Frozen protein arrays: a new method for arraying and detecting recombinant and native tissue proteins.

DNA microarrays are powerful tools for high throughput analysis of gene expression; however, they do not measure protein expression. Current methods for producing protein arrays require sophisticated equipment or extensive protein modification. We developed a low overhead, customizable assay platform called frozen protein arrays that can detect native proteins in protein lysates. Frozen protein arrays were formed from a block of frozen histologic embedding compound containing an array of wells. The wells were filled with samples, which freeze and bond to the block. Cryosections were cut and transferred to nitrocellulose-coated slides. The reproducibility, linearity, and sensitivity was confirmed using frozen protein arrays filled with prostate specific antigen. Frozen protein arrays could detect native tissue proteins. The alpha1 subunit of NaK-ATPase was detected in rat kidneys with a coefficient of variation of 4.3-6.6%. Frozen protein array analysis indicated that the protein abundance decreased by 48.7% following renal ischemia, similar to the 40% decrease by Western blotting. We conclude that frozen protein arrays are a low cost, moderate size platform for arraying samples including protein lysates. Production of many identical frozen protein arrays is easy, inexpensive, and requires only small sample volumes. The method is gentle on proteins as they remain frozen during production.

Animals↗
PubMed2002

Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions.

We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2003

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions.

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.

Binding Sites↗
PubMed2003

Profiling receptor tyrosine kinase activation by using Ab microarrays.

Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96- and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.

Animals↗
PubMed2002

Integrated approaches to therapeutic target gene discovery.

The identification of tractable drug targets is the first critical step in the long process of drug development, and the challenge for scientists in the post-genomic era is to couple gene and protein sequence information with biological insight to identify genes with the greatest therapeutic and commercial potential. An extraordinary array of genomics- and proteomics-based techniques is available for this endeavor, and combining multiple, complementary approaches enhances the informative power of such experimentation.

Animals↗
PubMed2003

Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer.

Tissue microarrays allow high throughput molecular profiling of diagnostic or predictive markers in cancer specimens and rapid validation of novel potential candidates identified from genomic and proteomic analyses in a large number of tumor samples. To validate the use of tissue microarray technology for all the main biomarkers routinely used to decide breast cancer prognostication and postsurgical adjuvant therapy, we constructed a tissue microarray from 97 breast tumors, with a single 0.6 mm core per specimen. Immunostaining of tissue microarray sections and conventional full sections of each tumor were performed using well-characterized prognostic markers (estrogen receptor ER, progesterone receptor PR and c-erbB2). The full section versus tissue microarray concordance for these stains was 97% for ER, 98% for PR, and 97% for c-erbB2, respectively, with a strong statistical association (kappa value more than 0.90). Fluorescence in situ hybridization analysis for HER-2/neu gene amplification from the single-core tissue microarray was technically successful in about 90% (87/97) of the cases, with a concordance of 95% compared with parallel analyses with the full sections. The correlation with other pathological parameters was not significantly different between full-section and array-based results. It is concluded that the constructed tissue microarray with a single core per specimen ensures full biological representativeness to identify the associations between biomarkers and clinicopathological parameters, with no significant associated sampling bias.

Adenocarcinoma↗
PubMed2003

Disease proteomics.

The sequencing of the human genome and that of numerous pathogens has opened the door for proteomics by providing a sequence-based framework for mining proteomes. As a result, there is intense interest in applying proteomics to foster a better understanding of disease processes, develop new biomarkers for diagnosis and early detection of disease, and accelerate drug development. This interest creates numerous opportunities as well as challenges to meet the needs for high sensitivity and high throughput required for disease-related investigations.

Disease↗
PubMed2003

Hydrogel-based protein microchips: manufacturing, properties, and applications.

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.

Equipment Design↗
PubMed2002

Cytokine assays.

Explore the source record for details and available documents.

Animals↗
PubMed2003

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins.

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.

Animals↗
PubMed2003

Antibody-based technologies for target discovery.

Antibodies have proven to be exquisite investigation tools in the field of life sciences. They also constitute one of the oldest and most successful biological products for diagnostics and therapeutics. This review investigates the current use of antibodies in target discovery. To address this topic in a larger context, established and emerging technologies that are expected to contribute to target discovery will first be examined. These technologies include: mass spectroscopic analysis of proteins, protein-protein interaction and other network analysis approaches, as well as protein and antibody arrays. The potential of antibody engineering and the ANTIBIOMIX technology will then be discussed; antibody therapeutics, however, will not be examined.

Animals↗