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

WordNet

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
the extent to which something is delayed or held back
lack of normal development of intellectual capacities (同)mental_retardation, backwardness, slowness, subnormality
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"
cut off the testicles (of male animals such as horses); "the vet gelded the young horse" (同)cut

PrepTutorEJDIC

(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
〈U〉遅延,遅滞 / 〈C〉遅らせるもの;妨害物 / 〈U〉〈C〉知能(学業など)の遅れ
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
〈動物,特に馬〉‘を'去勢する

Wikipedia preview

出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/02/18 11:20:06」(JST)

wiki en

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

UpToDate Contents

全文を閲覧するには購読必要です。 To read the full text you will need to subscribe.

1. ヘリコバクターピロリ感染に対する診断検査およびその適応 indications and diagnostic tests for helicobacter pylori infection
2. 点頭てんかんのマネージメントおよび予後 management and prognosis of infantile spasms
3. 尿路上皮（移行上皮）膀胱癌の検出のための尿中バイオマーカー urine biomarkers for the detection of urothelial transitional cell carcinoma of the bladder
4. 限局性初期乳癌における予後予測因子 prognostic and predictive factors in early non metastatic breast cancer
5. 後天性の凝固因子阻害物質 acquired inhibitors of coagulation

English Journal

Mechanisms of divergent effects of activated peroxisome proliferator-activated receptor-γ on mitochondrial citrate carrier expression in 3T3-L1 fibroblasts and mature adipocytes.

Bonofiglio D, Santoro A, Martello E, Vizza D, Rovito D, Cappello AR, Barone I, Giordano C, Panza S, Catalano S, Iacobazzi V, Dolce V, Andò S.SourceDept. Pharmacy, Health Sciences and Nutritional, University of Calabria 87036 Arcavacata di Rende (Cosenza), Italy.
Biochimica et biophysica acta.Biochim Biophys Acta.2013 Jun;1831(6):1027-36. doi: 10.1016/j.bbalip.2013.01.014. Epub 2013 Jan 28.
The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be es
PMID 23370576

Role of CC-chemokine receptor 5 on myocardial ischemia-reperfusion injury in rats.

Shen B, Li J, Gao L, Zhang J, Yang B.SourceDepartment of Cardiology, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan, 430060, People's Republic of China.
Molecular and cellular biochemistry.Mol Cell Biochem.2013 Jun;378(1-2):137-44. doi: 10.1007/s11010-013-1604-z. Epub 2013 Mar 3.
The expression level of CC-chemokine receptor 5 (CCR5) is enhanced post inflammatory stimulations and might play a crucial role on inflammatory cells infiltration post myocardial ischemia. The purpose of this study was to evaluate the role of CCR5 on myocardial ischemia-reperfusion (I/R) injury in r
PMID 23456481

Over-expression of Neuron-derived Orphan Receptor-1 (NOR-1) exacerbates neointimal hyperplasia after vascular injury.

Rodríguez-Calvo R, Guadall A, Calvayrac O, Navarro MA, Alonso J, Ferrán B, de Diego A, Muniesa P, Osada J, Rodríguez C, Martínez-González J.AbstractWe have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.
Human molecular genetics.Hum Mol Genet.2013 May 15;22(10):1949-59. doi: 10.1093/hmg/ddt042. Epub 2013 Feb 5.
We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The ai
PMID 23390133

Japanese Journal

Structure and Regulatory Expression of A Single Copy Alternative Oxidase Gene from the Yeast Pichia anomala

SAKAJO Shigeru,MINAGAWA Nobuko,YOSHIMOTO Akio
Bioscience, biotechnology, and biochemistry 63(11), 1889-1894, 1999-11-23
… By a gel mobility shift assay, a specific retardation band was detected when a protein extract from cells grown on an inducing carbon source was incubated with a UAS2-containing probe. …
NAID 110002679385

Receptor-Mediated Gene Transfer to Cells of Hepatic Origin by Galactosylated Albumin-Polylysine Complexes

HAN Junghee,LIM Mijung,YEOM Young Il
Biological & pharmaceutical bulletin 22(8), 836-840, 1999-08-15
… This vector was caracterized by dynamic laser light scattering, gel retardation assay, and electron microscopy to determine the particle size, electrostatic charge interaction, and 3-D structure. …
NAID 110003639770

Related Pictures

★リンクテーブル★

リンク元	「electrophoretic mobility shift assay」「gel shift analysis」「ゲル遅延度アッセイ」「mobility shift assay」
関連記事	「assay」「retardation」「g」