Abstract:
:New technologies are needed that can diagnose cancer more rapidly and accurately. These technologies must also have the ability to identify the particular cellular abnormalities contributing to the malignancy, thus directing the appropriate treatments. Such technologies should permit absolute quantitation of specific tumor biomarkers and their level of posttranslational modifications. Quantitative molecular profiling of cancer signaling networks would provide a more detailed understanding of the contribution of protein expression and posttranslational modification levels to tumorigenesis. We have developed a unique approach for absolute quantitation of protein expression that integrates affinity capture of proteolytic peptides with mass spectrometry and thus provides detection, identification, and quantitation of their cognate proteins. We have previously shown the high sensitivity and specificity of this approach. Here we demonstrate the absolute quantitation of a model peptide using our technology. We have used this approach to capture epitope-containing peptides from proteolytically digested target proteins, including p53, epidermal growth factor receptor (EGFR), and prostate-specific antigen (PSA). Our technology can easily be extended to the absolute quantitation of protein modification levels, in addition to the determination of protein expression levels, and can be readily adapted for use in a microarray format. This method offers an improved approach to protein chip technology that should prove useful for clinical diagnosis and drug development applications.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Warren EN,Jiang J,Parker CE,Borchers CHdoi
10.2144/05386su01subject
Has Abstractpub_date
2005-06-01 00:00:00pages
7-11eissn
0736-6205issn
1940-9818pii
05386SU01journal_volume
Supplpub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract::Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, a...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04361ST04
更新日期:2004-01-01 00:00:00
abstract::GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-ri...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114457
更新日期:2016-10-01 00:00:00
abstract::We describe here a simple and efficient transfection method for transient expression of cloned genes in cell lines and primary cultured cells. The method involves the use of DEAE-dextran to target DNA to the cellular endocytotic pathway and the use of a human adenovirus to ensure efficient lysis of endosomal vesicles....
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1994-08-01 00:00:00
abstract::The reliability of the PCR technique used to type two human variable number tandem repeats, that is, 3' to apolipoprotein B gene and locus D17S30, was examined using DNA samples of mixed human and microbial origin. Mixtures of human and microbial DNA were amplified, choosing microbes found commonly in the vagina. Tota...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-08-01 00:00:00
abstract::The PCR method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material (e.g., Roundup Ready Soybean and Bt-176 "Maximizer" Maize) in foodstuffs. The first step in the procedure, namely the purification of nucleic acids from the sample, is often the deciding factor ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/01312pf01
更新日期:2001-08-01 00:00:00
abstract::Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selec...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114537
更新日期:2017-04-01 00:00:00
abstract::Current protocols for screening proliferative factors for β cells ex vivo are time-consuming, require cell lines or dissociated islets, and often entail expensive specialized screening equipment. Here we present an efficient and lower cost alternative that utilizes intact mouse islets for the initial screening of prol...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114115
更新日期:2013-12-01 00:00:00
abstract::Thin spun-coat films (~4 nm thick) of graphene oxide (GO) constitute a versatile surface chemistry compatible with a broad range of technologically important sensor materials. Countless publications are dedicated to the nuances of surface chemistries that have been developed for sensors, with almost every material hav...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114188
更新日期:2014-07-01 00:00:00
abstract::Sensitive nucleic acid based detection methods such as in situ PCR, in situ RT-PCR and PRINS have great potential in the areas of developmental biology, pathogenesis and diagnostics. However, control of evaporation from in situ reactions is critical to ensure reliable data. Self-Seal Reagent, a component added directl...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97232pf01
更新日期:1997-08-01 00:00:00
abstract::The recently developed ultrasensitive reverse transcriptase (RT) test involving a PCR step can detect minute amounts of RT in single retroviral particles and is 10(6) times more sensitive than conventional RT assays. We have found that different DNA-dependent DNA polymerases like DNA Polymerase I from Escherichia coli...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1996-02-01 00:00:00
abstract::Bovine serum albumin, free of deoxyribonuclease activity, was obtained in our laboratory using ion-exchange chromatography followed by acetylation. Chromatography on four different resins (DEAE-52, P-11, hydroxylapatite and Q Sepharose fast-flow) was examined. Fractions from Q Sepharose chromatography, eluted with a l...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-11-01 00:00:00
abstract::The trend toward high-throughput techniques in molecular biology and the explosion of online scientific data threaten to overwhelm the ability of researchers to take full advantage of available information. This problem is particularly severe in the rapidly expanding area of gene expression experiments, for example, t...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/99276bc03
更新日期:1999-12-01 00:00:00
abstract::Here we report a simple new method for exposing cells to normoxic and hypoxic conditions using vacuum bags, normally employed for food storage, to establish and maintain low oxygen levels in vitro. Vacuum bags were gassed with a mixture containing specified levels of oxygen, then sealed, creating a hypoxic microenviro...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114341
更新日期:2015-10-01 00:00:00
abstract::A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguish...
journal_title:BioTechniques
pub_type:
doi:10.2144/03341dd09
更新日期:2003-01-01 00:00:00
abstract::A fully automated nucleic acid analysis system is described, which offers positive sample identification, improved sensitivity and reduced user interaction compared to conventional techniques. The system relies on the sequence-specific capture of DNA onto solid-phase particles, confirming product identity without the ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/99271pf01
更新日期:1999-07-01 00:00:00
abstract::Microarrays printed on glass slides are often constructed by covalently linking modified oligonucleotide probes to a derivatized surface at considerable expense. In this article, we demonstrate that 14-base oligonucleotides with a poly(T)10 - poly(C)10 tail (TC tag), but otherwise unmodified, can be linked by UV light...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112905
更新日期:2008-09-01 00:00:00
abstract::We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) into inhibitors of cell growth. The method is based on the use of budding yeast (Saccharomyces ce...
journal_title:BioTechniques
pub_type:
doi:10.2144/99274st08
更新日期:1999-10-01 00:00:00
abstract::We systematically evaluated the performance and reliability of several widely used, commercially available actin-filament probes in a highly motile breast adenocarcinoma cell line to optimize the visualization of F-actin-rich dynamic lamellipodia. We evaluated four Phalloidin-fluorophores, two anti-actin antibodies, a...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2018-0112
更新日期:2019-03-01 00:00:00
abstract::Recent advances in long reverse transcription (RT)-PCR technology allow the copying of full-length coding regions of large mRNAs in one step. Using long RT-PCR, one can be certain that a given cDNA is derived from a single mRNA. In what to our knowledge is a novel application, we can isolate and characterize splice va...
journal_title:BioTechniques
pub_type:
doi:10.2144/98252st01
更新日期:1998-08-01 00:00:00
abstract::prfectBLAST is a multiplatform graphical user interface (GUI) for the stand-alone BLAST+ suite of applications. It allows researchers to do nucleotide or amino acid sequence similarity searches against public (or user-customized) databases that are locally stored. It does not require any dependencies or installation a...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113953
更新日期:2012-11-01 00:00:00
abstract::A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern b...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1991-01-01 00:00:00
abstract::A modification of PCR-mediated gene synthesis strategy is introduced. This modification enables the synthesis of a gene from oligonucleotides comprising only one of the two strands. Bridging oligonucleotides (approximately 20-mers in length) complementary to the junctions of template strand oligonucleotides and two ou...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-03-01 00:00:00
abstract::Radiolabeled proteins are used in a variety of laboratory applications as well as in radioimmunotherapy. This review focuses on methods that utilize genetic engineering to introduce exogenous phosphorylation sites into proteins. Protein kinase substrate sites can be introduced into target proteins to serve as tags for...
journal_title:BioTechniques
pub_type: 杂志文章,评审
doi:
更新日期:2002-10-01 00:00:00
abstract::A complete image digitizing and processing system is described for capturing, enhancing and analyzing molecular fingerprints. The low-cost, high-resolution system features a Motorola 68000 processor, multi-tasking, a separate video coprocessor, and color or gray scale processing. Thousands of manipulations are possibl...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-10-01 00:00:00
abstract::Genome variation provides researchers with thousands of markers with which to study human demographic history and phenotypes. Insertion-deletion (indel) polymorphism is an important and abundant form of human genome variation, and convenient methods for genotyping indels are therefore needed. Here we evaluate dynamic ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/03352st01
更新日期:2003-08-01 00:00:00
abstract::We have designed and built a magnetic tweezers device that enables the application of calibrated stresses to soft materials while simultaneously measuring their microscale deformation using confocal microscopy. Unlike previous magnetic tweezers designs, our device is entirely portable, allowing easy use on microscopes...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113701
更新日期:2011-07-01 00:00:00
abstract::We describe a method using an inexpensive craft glue to routinely isolate specific areas of tissue as small as 1 mm2 from paraffin sections. The tissue may be digested to release nucleic acid suitable for PCR or reverse transcription PCR. The use of this procedure obviates the requirement for manual microdissection or...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/96205st04
更新日期:1996-05-01 00:00:00
abstract::We observed the presence of contaminating NADH oxidation activity in maltose binding protein (MBP) fusion proteins expressed in Escherichia coli and purified using conventional amylose resin-based affinity chromatography. This contaminating NADH oxidation activity was detectable with at least four different enzymes fr...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/0000113822
更新日期:2012-04-01 00:00:00
abstract::Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, p...
journal_title:BioTechniques
pub_type: 杂志文章,评审
doi:10.2144/00293bi01
更新日期:2000-09-01 00:00:00
abstract::In the design of oligonucleotide sequences for targeting DNA or RNA sequences, it can be difficult to identify sequences that will hybridize only to the intended target. The term "sequence-specific" or "sequence-nonspecific" is often used to describe the interactions of an oligonucleotide with a mixture of DNA or RNA....
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/96206pf01
更新日期:1996-06-01 00:00:00