Abstract：
:Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor membrane preparations that maintain the transmembrane signaling activity of the receptor and provide robust biophysical and biochemical assay systems. Here we report a technique for self-assembling functional beta2-adrenergic receptor (beta2AR) into a nanoscale phospholipid bilayer system (Nanodisc) that is highly soluble in aqueous solution. The approximately 10-nm nanobilayer particles contain beta2AR in a native-like phospholipid bilayer domain of approximately 100 phospholipid molecules circumferentially bound by a membrane scaffold protein (MSP). The resulting construct allows for access to the physiologically intracellular and extracellular faces of the receptor and thus allows unrestricted access of antagonists, agonists, and G proteins. These Nanodisc-solubilized GPCRs can be directly purified by normal chromatographic procedures. We define the resultant Nanodisc-embedded monomeric beta2AR by antagonist and agonist binding isotherms and demonstrate faithful G protein coupling.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Leitz AJ,Bayburt TH,Barnakov AN,Springer BA,Sligar SGdoi
10.2144/000112169subject
Has Abstractpub_date
2006-05-01 00:00:00pages
601-2, 604, 606, passimissue
5eissn
0736-6205issn
1940-9818pii
000112169journal_volume
40pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract：:Genomic analysis of circulating, cell-free DNA (cfDNA) is being used extensively for molecular diagnostics. Many approaches rely on the construction of cfDNA genomic libraries, targeted retrieval of specific genomic regions and analysis by next-generation DNA sequencing. Several steps during sample preparation require...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2019-0071
更新日期：2019-10-01 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：:RNA interference (RNAi) is a recent advance that provides the possibility to reduce the expression of specific target genes in cultured mammalian cells with potential applications on a genome-wide scale. However, to achieve this, robust methodologies that allow automated and efficient delivery of small interfering RNA...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04373RT01
更新日期：2004-09-01 00:00:00
abstract：:Here we describe an amplification method for global transcript analysis. The strategy relies on amplification of cDNA tags (signature tags) achieved by random fragmentation of the cDNAs to short tags of similar length, isolation of the 3' ends and then PCR amplification of the 3'-end signature tag population. This met...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04362ST02
更新日期：2004-02-01 00:00:00
abstract：:Here we describe a method for the isolation of PCR-ready genomic DNA from various zebrafish tissues that is based on a previously published murine protocol. The DNA solutions are of sufficient quality to allow PCR detection of transgenes from all commonly used zebrafish tissues. In sperm, transgene amplification was s...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112619
更新日期：2007-11-01 00:00:00
abstract：:Screening methods for chemotherapeutic agents usually rely on the cytotoxic properties of the drugs. However, agents that inhibit invasion may have more efficacy and cause fewer side effects. Various cellular invasion assays have been used to evaluate these types of compounds, including the modified Boyden chamber, mo...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/98246cr04
更新日期：1998-06-01 00:00:00
abstract：:Presepsin is a 13-kDa N-terminal glycoprotein of CD14. Previously, agitation-induced increases in presepsin levels have been reported; however, the mechanism remains poorly understood. In this study, we aimed to reveal the mechanism of presepsin increase. The agitated plasma or serum was separated using gel exclusion ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2020-0136
更新日期：2021-01-29 00:00:00
abstract：:A simple and rapid procedure for recovering the denaturing effect of methylmercuric hydroxide in agarose gel electrophoresis is described. The procedure consisted of the treatment of the commercial methylmercuric hydroxide solutions with Amberlite, a mixture of anion- and cation-exchange resins. This treatment greatly...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1992-04-01 00:00:00
abstract：:Address correspondence to Martin A. Kennedy, Department of Pathology & Carney Centre for Pharmacogenomics, University of Otago, Christchurch, PO Box 4345, Christchurch, New Zealand. E-mail: martin.kennedy@otago.ac.nz. ...
journal_title：BioTechniques
pub_type： 信件
doi：10.2144/000114481
更新日期：2016-12-01 00:00:00
abstract：:A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel t...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1992-05-01 00:00:00
abstract：:Chitin from crustacean shells has often been used to isolate and purify plant lectins that have an affinity for poly-N-acetylglucosamine (poly-GlcNAc). When we used washed chitin from crab shells as an affinity medium to isolate a lectin from Pinus strobus L. (eastern white pine) ovules, we found that a substance havi...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1992-02-01 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：:Immunofluorescence quantification of γH2AX foci is a powerful approach to quantify DNA double-strand breaks induced by cancer therapy or accidental exposure to ionizing radiation. Here we report a modification to the γH2AX immunofluorescence labeling method, whereby cells are stained in-solution before being spotted a...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000113738
更新日期：2011-09-01 00:00:00
abstract：:The transcription factor GATA-1 is a zinc finger DNA-binding protein essential for the development of red blood cells. When we expressed different regions of the zinc finger domain in bacteria using an isopropyl-beta-D-thiogalactoside (IPTG) inducible system, growth of bacteria harboring the active DNA-binding domain ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/19962004684
更新日期：1996-04-01 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：:The serum protein transferrin (Tf) is a valuable marker for genetic studies of primates, because it is usually polymorphic, exhibiting as many as 13 allelic forms with high heterozygosity. The standard procedure to detect the different phenotypes requires vertical electrophoresis on polyacrylamide gels for 18 h at 4 d...
journal_title：BioTechniques
pub_type：
doi：
更新日期：1993-11-01 00:00:00
abstract：:Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device optimization. The phase shift c...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112676
更新日期：2008-02-01 00:00:00
abstract：:To move microarray technology into the diagnostic realm, the impact of technical parameters, such as sample preparation and RNA extraction, needs to be understood and minimized. We evaluated the impact of two RNA extraction methods, DNase treatment and the amount of hybridized cDNA probe, on the outcome of microarray ...
journal_title：BioTechniques
pub_type：
doi：10.2144/03355st04
更新日期：2003-11-01 00:00:00
abstract：:Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports r...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1993-03-01 00:00:00
abstract：:Ribosome profiling is a powerful tool for characterizing in vivo protein translation at the genome scale, with multiple applications ranging from detailed molecular mechanisms to systems-level predictive modeling. Though highly effective, this intricate technique has yet to become widely used in the microbial research...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114302
更新日期：2015-06-01 00:00:00
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 ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/05386su01
更新日期：2005-06-01 00:00:00
abstract：:Sarah Webb explores how RNA sequencing innovations help scientists tackle the complexity of the transcriptome. ...
journal_title：BioTechniques
pub_type： 新闻
doi：10.2144/btn-2017-0105
更新日期：2018-02-01 00:00:00
abstract：:A new method has been developed to purify and detritylate milligram amounts of synthetic oligonucleotides. Dimethoxytrityl oligonucleotides from 15 to 100 nucleotides in length are applied in triethylammonium acetate or concentrated ammonium hydroxide to a disposable chromatographic cartridge, the NENSORB PREP Nucleic...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1990-04-01 00:00:00
abstract：:We have developed a novel vector pTCS, as a tool for efficient selection of open reading frame (ORF)-containing inserts. In pTCS clones containing an insert with an ORF a downstream marker gene (immE3, conferring resistance to colicin) is activated via translational coupling with the insert, and transformed cells can ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112629
更新日期：2007-12-01 00:00:00
abstract：:Quantitative PCR and reverse transcription PCR (RT-PCR) are widely used in biomedical, industrial and other research applications to determine the number of RNA or DNA molecules of a specific type and/or sequence in a sample of interest. We have developed an assay system to accurately quantitate PCR products that util...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1995-05-01 00:00:00
abstract：:We describe here a simple and rapid method for enzymatic DNA amplification using DNA template recovered from membrane filters previously used in hybridization analysis. This is done by first solubilizing membrane pieces carrying DNA of interest in dimethyl sulfoxide, followed by isopropanol precipitation and polymeras...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1993-04-01 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：:In this special section of BioTechniques, we examine the role of rapid molecular technologies in the detection and identification of agents of infectious disease (ID) and biological weapons (BWs). Besides the threat posed by the global proliferation of BW technologies, there are numerous emerging and reemerging ID age...
journal_title：BioTechniques
pub_type： 杂志文章,评审
doi：10.2144/03354ss01
更新日期：2003-10-01 00:00:00
abstract：:The microcomputer-based image analysis system IB-1000 (developed by Indiana Biotech, Highland, IN) for two-dimensional electrophoresis gels has been described previously (9). It allows the user to compare protein spots between two profiles and identify those spots that are commonly shared in both profiles. This report...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1989-04-01 00:00:00
abstract：:A head-to-tail trimer of the SV40 Bcl I-Bam H1 DNA fragment, specifying polyadenylation of RNA transcripts, was cloned as a cassette flanked by multiple restriction sites. Insertion of the trimer into several expression vectors efficiently prevented spurious expression of reporter genes resulting from transcriptional ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1989-03-01 00:00:00
