Name: oligopeptide competition assay for phosphorylation site determination
Uploaded: 2017-05-18T14:00:00
Description: Watch this Scientific Journal Video about Oligopeptide Competition Assay for Phosphorylation Site Determination at JoVE.com

Эта работа была поддержана грантом Национального исследовательского фонда Кореи, финансируемым правительством Кореи (MSIP) (№ 2015R1A2A1A10052663 и № 2014M1A3A3A02034698).

1. Безопасность ВНИМАНИЕ: Этот протокол использует [γ- 32 P] -ATP для оценки активности AMPK. Фосфор-32 является радиоактивным изотопом, в значительной степени испускающим бета-излучение. Поскольку размер бета-частицы чрезвычайно мал, он может легко проникать сквозь одежду...

<ol>
	<li>Hardie, D. G., Schaffer, B. E., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK:+An+energy-sensing+pathway+with+multiple+inputs+and+outputs.">AMPK: An energy-sensing pathway with multiple inputs and outputs.</a> Trends Cell Biol. 26, 190-201 (2016).</li><li>Gwinn, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK+phosphorylation+of+raptor+mediates+a+metabolic+checkpoint.">AMPK phosphorylation of raptor mediates a metabolic checkpoint.</a> Mol Cell. 30, 214-226 (2008).</li><li>Huang, H. C., Nguyen, T., Pickett, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Nrf2+at+Ser-40+by+protein+kinase+C+regulates+antioxidant+response+element-mediated+transcription.">Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.</a> J Biol Chem. 277, 42769-42774 (2002).</li><li>Niture, S. K., Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant-induced+modification+of+INrf2+cysteine+151+and+PKC-delta-mediated+phosphorylation+of+Nrf2+serine+40+are+both+required+for+stabilization+and+nuclear+translocation+of+Nrf2+and+increased+drug+resistance.">Antioxidant-induced modification of INrf2 cysteine 151 and PKC-delta-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance.</a> J Cell Sci. 122, 4452-4464 (2009).</li><li>Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GSK-3beta+acts+upstream+of+Fyn+kinase+in+regulation+of+nuclear+export+and+degradation+of+NF-E2+related+factor+2.">GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2.</a> J Biol Chem. 282, 16502-16510 (2007).</li><li>Steen, H., Jebanathirajah, J. A., Rush, J., Morrice, N., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+analysis+by+mass+spectrometry:+myths,+facts,+and+the+consequences+for+qualitative+and+quantitative+measurements.">Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.</a> Mol Cell Proteomics. 5, 172-181 (2006).</li><li>Dephoure, N., Gould, K. L., Gygi, S. P., Kellogg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+analysis+of+phosphorylation+sites:+a+quick+guide+for+cell+biologists.">Mapping and analysis of phosphorylation sites: a quick guide for cell biologists.</a> Mol Biol Cell. 24, 535-542 (2013).</li><li>Joo, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK+facilitates+nuclear+accumulation+of+Nrf2+by+phosphorylating+at+Serine+550.">AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at Serine 550.</a> Mol Cell Biol. 36, 1931-1942 (2016).</li><li>Sinclair, W. K., Holloway, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-lives+of+some+radioactive+isotopes.">Half-lives of some radioactive isotopes.</a> Nature. 167 (4244), 365 (1951).</li><li>Chang, B. Y., Chiang, M., Cartwright, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+Src+and+RACK1+is+enhanced+by+activation+of+protein+kinase+C+and+tyrosine+phosphorylation+of+RACK1.">The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1.</a> J Biol Chem. 276, 20346-20356 (2001).</li><li>Smith, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RACK1+interacts+with+filamin-A+to+regulate+plasma+membrane+levels+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.</a> Am J Physiol Cell Physiol. 305 (1), C111-C120 (2013).</li></ol>

В качестве простого и удобного способа оценки достоверности предсказанных сайтов фосфорилирования, опосредованных AMPK, здесь мы описываем киназный анализ in vitro , который может быть использован для обнаружения конкретного участка фосфорилирования с использованием конкурентных п?...

Фосфорилирование белка у конкретного остатка играет значительную роль в широком диапазоне клеточных процессов. Таким образом, понимание сигнальных сетей требует идентификации конкретных сайтов фосфорилирования. Кроме того, сайт фосфорилирования определяет влияние на функцию белка, поскольку отдельные домены в пределах белка обладают различными структурами и функциями. Активность фактора фактора 2 эритроидного фактора 2 (Nrf2) ядерного фактора, ключевого антиоксидантного транскрипционного фактора, регулируется двунаправленным образом посредством фосфорилирования в разных местах. Наши исследования были сосредоточены на киназах, которые катализируют фосфорилирование Nrf2. Реакция стресса Nrf2 против окислительного заражения происходит быстро, главным образом за счет фосфорилирования на серине 40 и опосредованного протеинкиназой C (PKC) -δ, которая активирует Nrf2 3,4 . Напротив, Fyn катализирует тормозное фосфорилирование Nrf2 при тирозине 568 для строгого контроля за деятельностью 5 . Наиболее распространенным методом, используемым для обнаружения сайтов фосфорилирования, является анализ методом жидкостной хроматографии / масс-спектрометрии (ЖХ / МС). Быстрые и высокочувствительные данные картирования сайта фосфорилирования могут быть получены таким образом; Однако он имеет несколько технических ограничений, часто генерирующих ложноотрицательные сигналы. Плохое покрытие последовательностей часто происходит при анализе ЖХ / МС. Для идентификации сайтов фосфорилирования требуется информация о максимальном покрытии белками аминокислот 6 . Протеолиз белка, представляющего интерес, с несколькими протеазами во время стадии пищеварения может помочь в улучшении охвата последовательностей. Другим препятствием для идентификации остатков фосфорилирования является легкая потеря фосфорной кислоты, которая часто наблюдается для сериновых и треонинфосфорилированных пептидов 6 , 7 . Лабильные фосфатные группы частоВысвобождается из фосфопептидов в процессе фрагментации 7 . Второй вариант при поиске сайтов фосфорилирования заключается в использовании пептидного микроматричного метода. Возможно проводить скрининг на целевые сайты киназ с использованием чипа микрочипов, содержащего пептидные фрагменты, полученные из представляющего интерес белка. Однако из-за требований к оборудованию для производства и обнаружения микрочипов, метод пептидных микроматриц считается отнимающим много времени и дорогостоящим. Для преодоления этих трудностей для протеинкиназы с известными мотивами распознавания может быть использован анализ in vitro -конкурирующих киназ in vitro . Если устанавливается мотив консенсусного распознавания киназы, предполагаемые сайты фосфорилирования субстрата-кандидата могут быть предсказаны, и аутентичность сайтов может быть подтверждена. Наиболее убедительным методом для этой процедуры является показать отмену фосфорилирования в мутантном белке, в котором предикатD остаток замещен нефосфорилируемой аминокислотой ( т.е. серин или треонин-аланин, тирозин-фенилаланин). Однако производство и выделение мутантных белков требует много времени. В качестве альтернативы на начальном этапе исследования конкурентный тест пептид-киназы является простым и удобным. Здесь мы опишем протокол для конкурентного анализа пептида in vitro и для проверки сайта фосфорилирования.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>208337</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>450243</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Calbiochem, Nottingham, UK</td>
			<td>539134</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1752</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPK</td>
			<td>Upstate Biotechnology, Lake Placid, NY</td>
			<td>14-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Nrf2 (WT)</td>
			<td>Abnova, Taipei City, Taiwan</td>
			<td>H00004780-P01</td>
			<td></td>
		</tr>
		<tr>
			<td>[&#947;-32P]-ATP</td>
			<td>PerkinElmer Life and Analytical Sciences, Waltham, MA</td>
			<td>NEG502A</td>
			<td></td>
		</tr>
		<tr>
			<td>EZblue staining reagent</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G1041</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu turbo DNA polymerase</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td>600250</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td>200415-51</td>
			<td>Avoid multiple thaw and freezing cycle</td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Duchefa Biochemie BV, Haarlem, Netherlands</td>
			<td>L1704</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix, Santa Clara, CA</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose LE</td>
			<td>iNtRON Biotechnology, Sungnam, South Korea</td>
			<td>32034</td>
			<td></td>
		</tr>
		<tr>
			<td>HiYield Plus Gel/PCR DNA Mini Kit</td>
			<td>Real Biotech Corporation, Taipei, Taiwan</td>
			<td>QDF100</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>161-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Bovogen Biologicals, Victoria, Australia</td>
			<td>BSA100</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose 4B</td>
			<td>GE Healthcare, Marlborough, MA</td>
			<td>17-0756-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid glacial</td>
			<td>Duksan pure chemicals, Ansan, South Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl alcohol</td>
			<td>Daejung Chemicals &#38; Metals, Siheung, South Korea</td>
			<td>5558-4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare, Marlborough, MA</td>
			<td>28-9558-09</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE kit</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>165-178</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>165-1746</td>
			<td></td>
		</tr>
		<tr>
			<td>Dancing shaker</td>
			<td>FINEPCR, Seoul, Korea</td>
			<td>CR300</td>
			<td>The machine is needed for washing step</td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator/shakers</td>
			<td>N-BIOTEK, GyeongGi-Do, Korea</td>
			<td>NB-205L</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuges</td>
			<td>LABOGENE, Seoul, Korea</td>
			<td>1730R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography columns</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>732-1010</td>
			<td></td>
		</tr>
	</tbody>
</table>

oligopeptide competition assay for phosphorylation site determination

Фосфорилирование белков в определенных местах определяет его конформацию и взаимодействие с другими молекулами. Таким образом, фосфорилирование белка влияет на биологические функции и характеристики клетки. В настоящее время наиболее распространенным методом обнаружения сайтов фосфорилирования является анализ методом жидкостной хроматографии / масс-спектрометрии (ЖХ / МС), быстрый и чувствительный метод. Однако относительно лабильные фосфатные фрагменты часто высвобождаются из фосфопептидов во время стадии фрагментации, что часто приводит к ложноотрицательным сигналам. В таких случаях традиционный анализ киназы in vitro с использованием сайт-направленных мутантов был бы более точным, но этот метод является трудоемким и трудоемким. Поэтому альтернативный способ с использованием пептидной конкуренции может быть предпочтительным. Установлен мотив консенсусного распознавания 5&#39;-аденозинмонофосфат-активированной протеинкиназы (AMPK) 1 и он был подтвержден с помощью библиотеки позиционного сканирующего пептида assAy 2 . Таким образом, сайты фосфорилирования AMPK для нового субстрата могут быть предсказаны и подтверждены с помощью конкурентного анализа пептидов. В этом отчете мы описываем подробные этапы и процедуры анализа in vitro- олигопептид-конкурирующих киназ, иллюстрируя AMPK-опосредованное фосфорилирование эритроидного фактора 2 (Nrf2), связанного с эритроидным фактором фактора II. Для аутентификации сайта фосфорилирования мы проводили последовательный анализ киназы in vitro с использованием сайт-специфического мутанта. В целом, конкурентный анализ пептидов дает способ скрининга нескольких потенциальных сайтов фосфорилирования и идентификации сайтов для валидации с помощью мутантов сайта фосфорилирования.

Рисунок 1 и Рисунок 2 демонстрируют результаты повторных экспериментов в ранее опубликованной статье 8 . Три различных олигопептида с 10 остатками, имитирующие предполагаемые сайты-мишени AMPK (№ 1, 148-157, включающие Ser153, №2, 330-339...

Watch this Scientific Journal Video about Oligopeptide Competition Assay for Phosphorylation Site Determination at JoVE.com

Oligopeptide Competition Assay for Phosphorylation Site Determination

Конкурентные тесты пептидов широко используются в различных молекулярных и иммунологических экспериментах. В этом документе описывается подробный метод анализа in vitro- олигопептид-конкурирующих киназ и соответствующие процедуры валидации, которые могут быть полезны для поиска специфических сайтов фосфорилирования.

Определение конкуренции на олигопептидах для определения местонахождения фосфорилирования

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

The overall goal of this procedure is to simultaneously assess multiple potential phosphorylation sites and to identify sites for subsequent validation by phosphorylation site mutants. This method can help answer key questions in the biochemistry and cell biology fields about the role of post-translational modifications in cell signaling. This technique allows easy budget friendly preliminary screening for phosphorylation sites and the protein of interest.The implication of this technique extended toward the diagnosis and therapy of diseases including inflammation and cancer because phosphorylation is an important biochemical event in signaling pathways in human diseases. Aiding the demonstration would be Sol-Bi Shin a graduate student from my laboratory who while perform site directed mutagenesis. First combine 50 nanograms of the plasma pGEX JST NRF2, 125 nanograms of forward primer, 125 nanograms of reverse primer, one microliter of DNTP mix 2.5 units of PFU DNA polymerase and 10x reaction buffer on ice.Add sterile distilled water to bring the volume to 50 microliters. Perform PCR to incorporate the mutant strand and then cool the sample on ice for two minutes. Add one microliter of DPNI restriction enzyme to the cooled sample.Mix the sample with gentle pipetting and then centrifuge the sample for one minute at 13, 000 times G.Incubate the sample at 37 degree Celsius for two hours to digest the parental double stranded DNA. Then toss supercompetent E.coli cells with endA1 and RACA1 mutations on ice. Add 50 microliters of the reaction mixture to the chilled cells and incubate on ice for 30 minutes.Meanwhile, preheat a heating block to 42 degree Celsius. Once incubation in complete, heat the mixture at 42 degree Celsius for 90 seconds and then cool the mixture on ice for two minutes. Add pre-warmed Lysogeny broth to the transformed cells.Incubate the mixture at 37 degree Celsius for one hour while shaking at 180 RPM. Then spread the cells on LB ampicillin agar plates and incubate the plates overnight at 37 degree Celsius. Transfer a single colony to five milliliters of Lysogeny broth with ampicillin.Incubate the samples overnight at 37 degree Celsius while shaking at 180 RPM. Extract the sample DNA using a standard mini prep kit for DNA sequence analysis. Run at least 200 nanograms of HDNA construct in a DNA analyzer to verify the mutant sequence.Express and purify the validated mutant NRF2 protein. Determine the quantity of mutated NRF2 produced before proceeding the kinase assay. To begin the peptide competition assay, prepare a 5X kinase buffer solution with an adenosine monophosphate concentration of 500 micromolar.Then thaw frozen samples of AMPK wild type human NRF2 and three oliogopeptides mimicking the AMPK phosphorylation sites of NRF2. For each peptide, while on ice, combine 0.43 milligrams of the peptide with 0.15 micrograms of AMPK, 0.4 micrograms of NRF2 and six microliters of 5X Kinase Buffer. Add sterile distilled water to achieve a final volume of 30 microliters.Then in a shielded area, set a heating block to 30 degree Celsius. Add one microcurie of gamma 32P ATP to each tube, mixing each solution thoroughly by pipetting up and down. Incubate the reaction mixtures at 30 degree Celsius for 15 to 30 minutes.During the incubation, prepare 7.5%STS PAGE gel. Then add three microliters of 10X STS sample buffer to each tube and mix thoroughly to stop the kinase reaction. Run the samples in the gel at 70 volts for 20 minutes and then at 140 volts for one hour.Upon completing the gel electrophoresis carefully remove the highly radioactive gel below the bromophenol blue line. Gently transfer the gel from the caster to a glass tube. Fix the gel in a solution of 50%methanol, 40%water and 10%acetic acid for 20 minutes.Then gently place the gel on filter paper and cover it with transparent wrap. Dry the gel with a vacuum gel dryer for one hour at 80 degree Celsius. Exposure the dry gel either to a phosphorus screen or an x-ray film for approximately 16 hours for two days respectively.Use a Phosphorimager to generate a high resolution TIFF or Bitmap file of the screen. To begin the AMPK activity assay thaw samples of AMPK and of mutant and wild type GST NRF2 on ice. Combine 0.4 micrograms of mutant GST NRF2 with 0.15 micrograms of AMPK and six microliters of 5X Kinase buffer.Add sterile distilled water to bring the volume to 30 microliters. Prepare another reaction mixture in this way with 0.4 micrograms of wild type GST NRF2. Add one microcurie of gamma 32P ATP to each reaction vial.Pipette the mixtures up and down several times and then briefly centrifuge the mixtures. Incubate the mixtures at 30 degree Celsius for 30 minutes in a shielded area and then terminate the kinase reaction with three microliters of 10X STS sample buffer. Run STS PAGE, fix the gel, and dry the gel using the same conditions as the competitive kinase assay.Expose the gel to a phosphorus screen overnight and scan the screen with the Phosphorimager. An in vitro AMPK assay was performed in the presence of three 10 residue oligopeptides mimicking AMPK phosphorylation sites on human NRF2. The oligopeptide mimicking the Serine 558 site offered the greatest competition for AMPK phosphorylation.An NRF2 mutant replacing Serine 558 with alanine was then synthesized and evaluated in an in vitro activity assay. Very little AMPK phosphorylation of the S558 A mutant was observed indicating that AMPK directly phosphorylates human NRF2 at Serine 558. Since peptides competitively bind to proteins the principle of this method may also be applied to identifying other post translation and modifications such as acetylization and lysine residues and protein protein interactions.Don't forget that working with radio isotope can be extremely hazardous. Radio isotope safety training should already be taken before performing this procedure.

oligopeptide-competition-assay-for-phosphorylation-site-determination

Research

JoVE Journal

Biochemistry

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

Высокая точность FRET на уровне одиночной молекулы для определения структуры биомолекулы

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

Assay для фосфорилирования и привязки микротрубочек наряду с локализацией тау белка в клетках рака прямой кишки

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Определение содержания гликогена в цианобактериях

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Идентификация Циклин зависимая киназа 1 сайты конкретных фосфорилирование киназы в Vitro пробирного

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

Определение функциональных белков регионов путем строительства химерных белка

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson&#039;s Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

Человеческая периферическая кровь Нейтрофилов Изоляция для допроса Паркинсона Связанные LRRK2 Киназы Путь путем оценки Rab10 фосфорилирования

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Сайт Направленный мутагенез для In Vitro и In Vivo экспериментов примером взаимодействия РНК в Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H+- and O2- sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

Использование внеклеточного анализатора flux для измерения изменений в гликолизе и окислительной фосфорилации во время емки спермы мыши

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Acyl-PEGyl Обмен Гель Сдвиг Анализ для количественного определения Palmitoylation мозга Membrane белков

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Нерадиоактивный анализ для измерения полинуклеотида фосфорилирования малых нуклеотидных субстратов

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...