関: electrophoretic mobility shift assay、gel mobility shift assay、gel shift analysis

WordNet

use a shift key on a keyboard; "She could not shift so all her letters are written in lower case"
the time period during which you are at work (同)work shift, duty period
the act of moving from one place to another; "his constant shifting disrupted the class" (同)shifting
an event in which something is displaced without rotation (同)displacement
a crew of workers who work for a specific period of time
change place or direction; "Shift ones position" (同)dislodge, reposition
change gears; "you have to shift when you go down a steep hill"
change in quality; "His tone shifted"
change phonetically as part of a systematic historical change; "Grimm showed how the consonants shifted"
move and exchange for another; "shift the date for our class reunion"
move from one setting or context to another; "shift the emphasis"; "shift ones attention"
a quantitative or qualitative test of a substance (especially an ore or a drug) to determine its components; frequently used to test for the presence or concentration of infectious agents or antibodies etc.
analyze (chemical substances)
a substance that is undergoing an analysis of its components
an appraisal of the state of affairs; "they made an assay of the contents"; "a check on its dependability under stress" (同)check
a written report of the results of an analysis of the composition of some substance
continuously varying; "taffeta with shifting colors"
changing position or direction; "he drifted into the shifting crowd"; "their nervous shifting glances"; "shifty winds" (同)shifty
(of soil) unstable; "shifting sands"; "unfirm earth" (同)unfirm
hardy and sure-footed animal smaller and with longer ears than the horse
a pompous fool
a colloid in a more solid form than a sol (同)colloidal gel
become a gel; "The solid, when heated, gelled"

PrepTutorEJDIC

〈人・場所・位置など〉‘を'『移し替える』,置き換える / …‘を'変える,取り替える / 〈自動車のギヤ〉‘を'入れ変える / (…へ)『変わる』,移る《+『to』+『名』》 / 車のギヤを変える;〈車が〉ギヤが入れ変わる・『変換』,転換;移動 / (仕事、作業の)『交替』,交替製;交替の組(人);交替時間 / (特に,自動車)変速装置 / やりくり[算段],一時しのぎの手段,便法;ずるい手段,策略 / シフトドレス(肩から腰までまっすぐした線のゆったりしたワンピース);スリップ
(金属の品質・鉱石の金属含有量・薬品の)分析,試金 / 試金物,分析物 / …'を'試金する,分析する
ロバ / ばか者
《米俗》しり,けつ(《英俗》arse)
ゲル(コロイド溶液の凝固した状態;ゼラチン・寒天など) / =jell

Wikipedia preview

出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2016/06/14 05:10:27」(JST)

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Lane 1 is a negative control, and contains only genetic material. Lane 2 contains protein as well as a DNA fragment that, based on its sequence, does not interact. Lane 3 contains protein and a DNA fragment that does react; the resulting complex is larger, heavier, and slower-moving. The pattern shown in lane 3 is the one that would result if all the DNA were bound and no dissociation of complex occurred during electrophoresis. When these conditions are not met a second band might be seen in lane 3 reflecting the presence of free DNA or the dissociation of the DNA-protein complex.

An electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common affinity electrophoresis technique used to study protein–DNA or protein–RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation, DNA replication, DNA repair or RNA processing and maturation. Although precursors can be found in earlier literature, most current assays are based on methods described by Garner and Revzin ^[1] and Fried and Crothers.^[2]

Principle

A mobility shift assay is electrophoretic separation of a protein–DNA or protein–RNA mixture on a polyacrylamide or agarose gel for a short period (about 1.5-2 hr for a 15- to 20-cm gel).^[3] The speed at which different molecules (and combinations thereof) move through the gel is determined by their size and charge, and to a lesser extent, their shape (see gel electrophoresis). The control lane (DNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. However, assuming that the protein is capable of binding to the fragment, the lane with protein present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to protein which is 'shifted' up on the gel (since it has moved more slowly).

Under the correct experimental conditions, the interaction between the DNA (or RNA) and protein is stabilized and the ratio of bound to unbound nucleic acid on the gel reflects the fraction of free and bound probe molecules as the binding reaction enters the gel. This stability is in part due to a "caging effect", in that the protein, surrounded by the gel matrix, is unable to diffuse away from the probe before they recombine.^[4] If the starting concentrations of protein and probe are known, and if the stoichiometry of the complex is known, the apparent affinity of the protein for the nucleic acid sequence may be determined.^[5] Unless the complex is very long lived under gel conditions, or dissociation during electrophoresis is taken into account, the number derived is an apparent Kd. If the protein concentration is not known but the complex stoichiometry is, the protein concentration can be determined by increasing the concentration of DNA probe until further increments do not increase the fraction of protein bound. By comparison with a set of standard dilutions of free probe run on the same gel, the number of moles of protein can be calculated.^[3]

An antibody that recognizes the protein can be added to this mixture to create an even larger complex with a greater shift. This method is referred to as a supershift assay, and is used to unambiguously identify a protein present in the protein – nucleic acid complex.

Often, an extra lane is run with a competitor oligonucleotide to determine the most favorable binding sequence for the binding protein. The use of different oligonucleotides of defined sequence allows the identification of the precise binding site by competition (not shown in diagram). Variants of the competition assay are useful for measuring the specificity of binding and for measurement of association and dissociation kinetics.

Once DNA-protein binding is determined in vitro, a number of in silico algorithms can narrow the search for identification of the transcription factor. Consensus sequence oligonucleotides for the transcription factor of interest will be able to compete for the binding, eliminating the shifted band, and must be confirmed by supershift. If the predicted consensus sequence fails to compete for binding, identification of the transcription factor may be aided by Multiplexed Competitor EMSA (MC-EMSA), whereby large sets of consensus sequences are multiplexed in each reaction, and where one set competes for binding, the individual consensus sequences from this set are run in a further reaction.^[6]

For visualization purposes, the nucleic acid fragment is usually labelled with a radioactive, fluorescent or biotin label. Standard ethidium bromide staining is less sensitive than these methods and can lack the sensitivity to detect the nucleic acid if small amounts of nucleic acid or single-stranded nucleic acid(s) are used in these experiments. When using a biotin label, streptavidin conjugated to an enzyme such as horseradish peroxidase is used to detect the DNA fragment (Non-radioactive EMSA review). While isotopic DNA labeling has little or no effect on protein binding affinity, use of non-isotopic labels including flurophores or biotin can alter the affinity and/or stoichiometry of the protein interaction of interest. Competition between fluorophore- or biotin-labeled probe and unlabeled DNA of the same sequence can be used to determine whether the label alters binding affinity or stoichiometry.

References

^ Garner MM, Revzin A (July 1981). "A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system". Nucleic Acids Res. 9 (13): 3047–60. doi:10.1093/nar/9.13.3047. PMC 327330. PMID 6269071.
^ Fried M, Crothers DM (December 1981). "Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis". Nucleic Acids Res. 9 (23): 6505–25. doi:10.1093/nar/9.23.6505. PMC 327619. PMID 6275366.
^ ^a ^b Ausubel, Frederick M. (1994). Current Protocols in molecular biology. Chichester: John Wiley & Sons. pp. 12.2.1–11. ISBN 0-471-50337-1.
^ Fried MG, Liu G. (1994). "Molecular sequestration stabilizes CAP- DNA complexes during polyacrylamide gel electrophoresis". Nucleic Acids Research 22(23): 5054-5059. doi: 10.1093/nar/22.23.5054. PMID 7800499.
^ Fried MG (1989) "Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay". Electrophoresis 10, 366-376. PMID 2670548.
^ Smith AJ, Humphries SE (January 2009). "Characterization of DNA-binding proteins using multiplexed competitor EMSA". J. Mol. Biol. 385 (3): 714–7. doi:10.1016/j.jmb.2008.11.035. PMID 19059416.

Protocols

Dr. Mirmira EMSA Protocol
Preparation of nuclear extract for EMSA
Jonathan Flint Lab Protocol
EMSA for Transcription Factor Binding
Chemiluminescent Gel Shift Protocol

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Japanese Journal

Direct interaction between EFL1 and SBDS is mediated by an intrinsically disordered insertion domain

Asano Nozomi,Atsuumi Haruka,Nakamura Akiyoshi,Tanaka Yoshikazu,Tanaka Isao,Yao Min
Biochemical and Biophysical Research Communications 443(4), 1251-1256, 2014-01-24
… In the present study, the interaction between EFL1 and SBDS was analyzed by size exclusion chromatography, gel shift assay, and isothermal titration calorimetry (ITC). …
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Dengue Envelope Domain Ⅲ-Peptide Binding Analysis via Tryptophan Fluorescence Quenching Assay

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サーマルシフトアッセイを用いた魚肉アクトミオシンの熱変性過程における疎水性構造の動態解析

矢田雄也,石田貴之,久保田光俊,増田太郎,豊原治彦
日本水産学会誌 80(4), 594-600, 2014
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リンク元	「ゲルシフト解析」「electrophoretic mobility shift assay」「gel shift analysis」「ゲルシフト法」「gel mobility shift assay」
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