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
the quality of moving freely

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)
可動性,流動性,変わりやすさ;(軍隊の)機動力

Wikipedia preview

出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2014/05/11 17:03:43」(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 (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

UpToDate Contents

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English Journal

Interaction of cytochrome c with tRNA and other polynucleotides.

Suryanarayana T, Uppala JK, Garapati UK.SourceDepartment of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, 500046, India, tssl@uohyd.ernet.in.
Molecular biology reports.Mol Biol Rep.2012 Jun 22. [Epub ahead of print]
Interaction between tRNA and other polynucleotides with cytochrome c was studied by visible spectroscopy, fluorescence spectroscopy and gel mobility shift assay in view of the recently reported important regulatory role of tRNA in cytochrome c mediated apoptotic pathway. Visible spectroscopy showed
PMID 22722995

Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A*.

Akter F, Mie M, Grimm S, Nygren PÅ, Kobatake E.SourceDepartment of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology , 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Analytical chemistry.Anal Chem.2012 Jun 5;84(11):5040-6. Epub 2012 May 16.
The chemical reactions used to make antibody-DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and
PMID 22571843

Japanese Journal

Oligomerization and DNA-Binding Capacity of Pmr, a Histone-Like Protein H1 (H-NS) Family Protein Encoded on IncP-7 Carbazole-Degradative Plasmid pCAR1

SUZUKI Chiho,YUN Choong-Soo,UMEDA Takashi,TERABAYASHI Tsuguno,WATANABE Kazuya,YAMANE Hisakazu,NOJIRI Hideaki
Bioscience, biotechnology, and biochemistry 75(4), 711-717, 2011-04-23
… To clarify the mode of function of Pmr, we performed gel filtration chromatography analysis and protein-protein cross-linking, and found that Pmr forms homo-oligomers, consisting of its homodimers. … Additionally, evaluating protein-DNA affinity is important to clarify the mode of function of Pmr, and hence we performed an electrophoretic mobility shift assay. …
NAID 10028271550

The Response Regulator RpaB Binds to the Upstream Element of Photosystem I Genes To Work for Positive Regulation under Low-Light Conditions in Synechocystis sp Strain PCC 6803

日原由香子
JOURNAL OF BACTERIOLOGY 191(5), 1581-1586, 2009-3
… The upstream element enhances the basal promoter activity under low-light conditions, whereas this positive regulation is lost immediately after the shift to high-light conditions. … A gel mobility shift assay revealed that a response regulator RpaB binds to the HLR1 sequence in PSI promoters. …
NAID 120001658776

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リンク元	「ゲルシフト解析」「gel shift assay」「gel shift analysis」「ゲル移動度シフト法」「ゲル移動度シフトアッセイ」
関連記事	「shift」「assay」「mobility」「g」