وأيد هذا العمل من قبل المعاهد الوطنية للصحة / NIDCR المنح 1R01DE019796.

<ol>
	<li>Bender, M., Kemp, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-18+Studies+of+the+Mechanism+of+the+alpha-Chymotrypsin-catalyzed+Hydrolysis+of+Esters.">Oxygen-18 Studies of the Mechanism of the alpha-Chymotrypsin-catalyzed Hydrolysis of Esters.</a> Journal of the American Chemical Society. 79, 111-116 (1957).</li><li>Sharon, N., Grisaro, V., Neumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pepsin-catalyzed+exchange+of+oxygen+atoms+between+water+and+carboxylic+acids.">Pepsin-catalyzed exchange of oxygen atoms between water and carboxylic acids.</a> Archives of Biochemistry and Biophysics. 97, 219-221 (1962).</li><li>Antonov, V., Ginodman, L., Rumsh, L., Kapitannikov, Y., Barshevskaya, T., Yavashev, L., Gurova, A., Volkova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanisms+of+action+of+proteolytic+enzymes+using+heavy+oxygen+exchange.">Studies on the mechanisms of action of proteolytic enzymes using heavy oxygen exchange.</a> European Journal of Biochemistry. 117, 195-200 (1981).</li><li>Fenselau, C., Yao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18O2-labeling+in+quantitative+proteomic+strategies:+a+status+report.">18O2-labeling in quantitative proteomic strategies: a status report.</a> Journal of Proteome Research. 8, 2140-2143 (2009).</li><li>Miyagi, M., Rao, K. C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+18O-labeling+strategies+for+quantitative+proteomics.">Proteolytic 18O-labeling strategies for quantitative proteomics.</a> Mass Spectrom. Rev. 26, 121-136 (2007).</li><li>Ye, X., Luke, B., Andresson, T., Blonder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18O+stable+isotope+labeling+in+MS-based+proteomics.">18O stable isotope labeling in MS-based proteomics.</a> Brief Funct. Genomic Proteomic. 8, 136-144 (2009).</li><li>Bantscheff, M., Schirle, M., Sweetman, G., Rick, J., Kuster, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry+in+proteomics:+a+critical+review.">Quantitative mass spectrometry in proteomics: a critical review.</a> Analytical and Bioanalytical Chemistry. 389, 1017-1031 (2007).</li><li>Yao, X., Afonso, C., Fenselau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+proteolytic+18O+labeling:+endoprotease-catalyzed+16O-to-18O+exchange+of+truncated+peptide+substrates.">Dissection of proteolytic 18O labeling: endoprotease-catalyzed 16O-to-18O exchange of truncated peptide substrates.</a> Journal of Proteome Research. 2, 147-152 (2003).</li><li>Robinson, S., Niles, R. K., Witkowska, H. E., Rittenbach, K. J., Nichols, R. J., Sargent, J. A., Dixon, S. E., Prakobphol, A., Hall, S. C., Fisher, S. J., Hardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mass+spectrometry-based+strategy+for+detecting+and+characterizing+endogenous+proteinase+activities+in+complex+biological+samples.">A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samples.</a> Proteomics. 8, 435-445 (2008).</li><li>Cottrell, G. S., Padilla, B. E., Amadesi, S., Poole, D. P., Murphy, J. E., Hardt, M., Roosterman, D., Steinhoff, M., Bunnett, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomal+endothelin-converting+enzyme-1:+a+regulator+of+beta-arrestin-dependent+ERK+signaling.">Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling.</a> The Journal of Biological Chemistry. 284, 22411-22425 (2009).</li><li>Subramanian, S., Hardt, M., Choe, Y., Niles, R. K., Johansen, E. B., Legac, J., Gut, J., Kerr, I. D., Craik, C. S., Rosenthal, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemoglobin+cleavage+site-specificity+of+the+Plasmodium+falciparum+cysteine+proteases+falcipain-2+and+falcipain-3.">Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.</a> PLoS ONE. 4, e5156 (2009).</li><li>Mason, C., Therneau, T., Eckel-Passow, J., Johnson, K., Oberg, A., Olson, J., Nair, K., Muddiman, D. C., Bergen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+automatically+interpreting+mass+spectra+of+18O-labeled+isotopic+clusters.">A method for automatically interpreting mass spectra of 18O-labeled isotopic clusters.</a> Molecular &amp; Cellular Proteomics. 6, 305-318 (2007).</li><li>Hicks, W. A., Halligan, B. D., Slyper, R. Y., Twigger, S. N., Greene, A. S., Olivier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+and+identification+using+18O+labeling+with+an+ion+trap+mass+spectrometer+and+the+analysis+software+application+"ZoomQuant&amp;quot.">Simultaneous quantification and identification using 18O labeling with an ion trap mass spectrometer and the analysis software application "ZoomQuant&amp;quot.</a> J. Am. Soc. Mass Spectrom. 16, 916-925 (2005).</li><li>Qian, W. -. J., Petritis, B. O., Nicora, C. D., Smith, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-catalyzed+oxygen-18+labeling+for+quantitative+proteomics.">Trypsin-catalyzed oxygen-18 labeling for quantitative proteomics.</a> Methods Mol. Biol. 753, 43-54 (2011).</li><li>Ye, X., Luke, B. T., Johann, D. J., Ono, A., Prieto, D. A., Chan, K. C., Issaq, H. J., Veenstra, T. D., Blonder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+method+for+computing+(18)O/(16)O+ratios+of+differentially+stable-isotope+labeled+peptides+in+the+context+of+postdigestion+(18)O+exchange/labeling.">Optimized method for computing (18)O/(16)O ratios of differentially stable-isotope labeled peptides in the context of postdigestion (18)O exchange/labeling.</a> Anal. Chem. 82 (18), 5878-5886 (2010).</li><li>Hardt, M., Lam, D. K., Dolan, J. C., Schmidt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+proteolytic+processes+in+human+cancer+microenvironments+by+microdialysis+and+activity-based+mass+spectrometry.">Surveying proteolytic processes in human cancer microenvironments by microdialysis and activity-based mass spectrometry.</a> Proteomics Clin. Appl. 5, 636-643 (2011).</li><li>Gattiker, A., Bienvenut, W., Bairoch, A., Gasteiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FindPept,+a+tool+to+identify+unmatched+masses+in+peptide+mass+fingerprinting+protein+identification.">FindPept, a tool to identify unmatched masses in peptide mass fingerprinting protein identification.</a> Proteomics. 2, 1435-1444 (2002).</li><li>Shevchenko, A., Chernushevich, I., Ens, W., Standing, K. G., Thomson, B., Wilm, M., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+'de+novo'+peptide+sequencing+by+a+combination+of+nanoelectrospray,+isotopic+labeling+and+a+quadrupole/time-of-flight+mass+spectrometer.">Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer.</a> Rapid Commun. Mass Spectrom. 11, 1015-1024 (1997).</li><li>Niles, R. K., Witkowska, H. E., Allen, S., Hall, S. C., Fisher, S. J., Hardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-catalyzed+oxygen-18+labeling+of+peptides.">Acid-catalyzed oxygen-18 labeling of peptides.</a> Analytical Chemistry. 81, 2804-2809 (2009).</li><li>Bender, M., Stone, R., Dewey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+Isotopic+Oxygen+Exchange+between+Substituted+Benzoic+Acids+and+Water.">Kinetics of Isotopic Oxygen Exchange between Substituted Benzoic Acids and Water.</a> Journal of the American Chemical Society. 78, 319-321 (1956).</li><li>Kuster, B., Schirle, M., Mallick, P., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scoring+proteomes+with+proteotypic+peptide+probes.">Scoring proteomes with proteotypic peptide probes.</a> Nature Reviews Molecular Cell Biology. 6, 577-583 (2005).</li></ol>

الإعلان عن أي تضارب في المصالح.

من خلال الجمع بين النظائر المستقرة وضع العلامات وعالية الدقة قياس الطيف الكتلي بطريقة حلها في الوقت، وطريقة باليو-TimeCourse يسمح لتحليل ديناميكية من الجيل من المنتجات الببتيد. ويمكن استخدام الفحص لتوليد مستقرة الببتيدات المسمى isotopically للدراسات البروتيوميات الكمية وال?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم المادة </td><td> شركة </td><td> كتالوج رقم </td></tr><tr><td> أعمدة PepClean C-18 سبين </td><td> الحرارية </td><td> 89870 </td></tr><tr><td> OPTI-TOF MALDI الهدف 384 لوحة </td><td> AB SCIEX </td><td> 1016629 </td></tr><tr><td> 4800 MALDI TOF / TOF </td><td> AB SCIEX </td><td></td></tr></tbody></table> الجدول 1. مواد <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td></tr><tr><td> ألفا cyano-4-hydroxycinnamic حمض </td><td> سيغما الدريتش </td><td> 70990-1G-F </td></tr><tr><td> ألبومين المصل البقري (BSA) </td><td> سيغما الدريتش </td><td> A3294-10G </td></tr><tr><td> Dithiothreitol (DTT) </td><td&gt; أكروس </td><td> 16568-0050 </td></tr><tr><td> Iodoacetamide (IAM) </td><td> سيغما الدريتش </td><td> 1149-5G </td></tr><tr><td> Endothelin تحويل انزيم-1 (ECE-1) </td><td> R &amp; D أنظمة </td><td> 1784-ZN </td></tr><tr><td> التربسين الذهب </td><td> Promega </td><td> V5280 </td></tr><tr><td> O-18 المياه، 97٪ ذرة 18 O </td><td> سيغما الدريتش </td><td> 329878-1G </td></tr><tr><td> Trifluoroacetic حمض (TFA) </td><td> الحرارية </td><td> 28904 </td></tr><tr><td> الشامل لمعايير عدة معايرة أدوات AB TOF / TOF SCIEX </td><td> AB SCIEX </td><td> 4333604 </td></tr></tbody></table> الجدول 2. الكواشف

protease- and acid-catalyzed labeling workflows employing 18o-enriched water

النظائر المستقرة هي أدوات أساسية في مطياف الكتلة البيولوجية. تاريخيا، تم 18 O-النظائر المستقرة المستخدمة على نطاق واسع لدراسة آليات الحفاز من الإنزيمات المحللة للبروتين 1-3. مع ظهور البروتيوميات الطيف القائم الشامل، أصبح إدماج إنزيمي المحفزة من 18 ذرات من O-مستقر الماء المخصب isotopically وسيلة شعبية لمقارنة مستويات بروتين تعبير كميا (مراجعتها من قبل Fenselau وياو 4، مياجي وراو 5 و يي وآخرون 6). 18 O وضع العلامات تشكل بديلا بسيطة ومنخفضة التكلفة للمواد الكيميائية (مثل iTRAQ، ICAT) والتمثيل الغذائي تقنيات الوسم (على سبيل المثال SILAC) 7. اعتمادا على الأنزيم البروتيني المستخدمة، يمكن وضع العلامات 18 O يؤدي إلى إدماج تصل إلى ذرات منهما 18-O في المجموعة C-الكربوكسيل محطة للمنتج الانقسام 3 </sup&gt;. ويمكن تقسيم رد الفعل العلامات إلى قسمين عمليات مستقلة، والانقسام ببتيد السندات وتبادل الأكسجين الكربوكسيل رد فعل 8. لدينا في باليو (ع rotease-A abeling ل ه ssisted mploying 18 O التخصيب الماء) تكييف الأنزيمية 18 O وضع العلامات، ونحن تستخدم 50٪ 18 O-المخصب لانتاج المياه التوقيعات النظائر مميزة. بالاشتراك مع عالية الدقة بمساعدة الليزر مصفوفة الامتزاز التأين وقت من رحلة مطياف الكتلة جنبا إلى جنب (MALDI-TOF/TOF MS / MS)، يمكن استخدام المغلفات النظائر لتحديد المنتجات المميزة الانقسام مع مستوى عال من الدقة. ونحن قد استخدمت سابقا باليو-منهجية لكشف وتميز البروتياز الذاتية 9 و رصد ردود بروتين 10-11. منذ باليو بترميز جوهر رد الفعل الانقسام بروتين، والإعداد التجريبية هو enri بسيطة والكيمياء الحيويةيمكن الخطوات chment من المنتجات الانقسام التحايل. يمكن بسهولة باليو-الأسلوب أن تمتد إلى (ط) التجارب بالطبع الوقت الذي رصد لديناميات تفاعلات الانقسام وبروتين (ب) تحليل العينات البيولوجية في التحلل البروتيني المعقد الذي يمثل الظروف الفسيولوجية. باليو-TimeCourse تجارب مساعدة في تحديد خطوات المعالجة معدل منظم للتفاعل وسيطة في مجمع ردود الفعل مسار بروتين. وعلاوة على ذلك، فإن رد فعل باليو تتيح لنا التعرف الإنزيمات المحللة للبروتين مثل التربسين سيرين البروتياز قادر إلى rebind المنتجات الانقسام وتحفيز إدماج في 18 2 O-ذرية. ويمكن استخدام مثل هذه &quot;انقر نقرا مزدوجا وضع العلامات&quot; الانزيمات لpostdigestion 18 O وضع العلامات، والتي وصفت حصرا الببتيدات عن طريق تفاعل الأوكسجين الصرف الكربوكسيل. استراتيجية الثالث من عمرنا يمتد وصفها توظيف 18 O التخصيب خارج المياه الانزيمات ويستخدم الرقم الهيدروجيني الحمضية الظروف لإدخال 18 علامة النظائر مستقرة O-atures في الببتيدات.

استخدمنا سير العمل باليو-TimeCourse لرصد حيوي إدماج نظائر مستقرة O-18 في المنتجات الانقسام الببتيد التي تم إنشاؤها بواسطة الإنزيمات المحللة للبروتين. النهج قدم هو أداة مرنة لدراسة مسارات المعالجة نسبيا لبروتين الركيزة مختلفة وتركيبات البروتيني. ردود الفعل من قبل أخ...

Watch this Scientific Journal Video about Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water at JoVE.com

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

وضع العلامات النظائر المستقرة العمل توظيف<sup&gt; 18</sup&gt; O-المخصب المياه (LEO-سير العمل) هي أدوات متعددة للدراسات البروتيوميات الكمية والنوعية. في العمل بمساعدة الأنزيم البروتيني (باليو)،<sup&gt; 18</supيتم إدخال&gt; O-ذرات من الانقسام والكربوكسيل بروتين تفاعلات تبادل الأكسجين بوساطة البروتياز. في سير العمل (اليو) حمض المحفزة،<sup&gt; 18</supيتم إدخال&gt; O-ذرات الأكسجين عن طريق تبادل الكربوكسيل في انخفاض الرقم الهيدروجيني.

مهام سير العمل صفها البروتيني وحمض حفز توظيف-<sup&gt; 18</sup&gt; O-المخصب المياه

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

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Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

11.9K Views.  Boston Biomedical Research Institute. وضع العلامات النظائر المستقرة العمل توظيف 18 O-المخصب المياه (LEO-سير العمل) هي أدوات متعددة للدراسات البروتيوميات الكمية والنوعية. في العمل بمساعدة الأنزيم البروتيني (باليو)، 18 O-ذرات من الانقسام والكربوكسيل بروتين تفاعلات تبادل الأكسج...

Video: Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

قياس إيماس في الخلايا العصبية عن طريق وصفها وزير الخارجية

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

موريس تجربة المتاهة المائية

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, <a href="http://jove.com/index/details.stp?ID=910">click here</a>

Sigma&#039;s Non-specific Protease Activity Assay - Casein as a Substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

سيجما للنشاط غير محددة البروتياز الفحص -- باعتباره الركيزة الكازين

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

بطاقات المواد الفلورية ذبابة الفاكهة</emهياكل القلب>

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

استخدام أدوات الكشف البروتياز نيون تحديد نشاط الأنزيم البروتيني

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

GC القائم على الكشف عن حمض Aldononitrile خلات الجلوكوزامين والموراميك Derivatized لتحديد بقايا الميكروبي في التربة

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

الماوس جزيرة من العزلة انجرهانز باستخدام مزيج من كولاجيناز تنقية والبروتياز متعادلة

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

تحديد غشاء طوبولوجيا البروتين عن طريق الإسفار البروتياز الحماية (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

سير عمل TEM وتحليل النتائج وإدارة البيانات باستخدام نهج رؤية الآلة

A Machine-Vision Approach to Transmission Electron Microscopy Workflows, Results Analysis and Data Management

Transmission electron microscopy (TEM) enables users to study materials at their fundamental, atomic scale. Complex experiments routinely generate thousands of images with numerous parameters that require time-consuming and complicated analysis. AXON synchronicity is a machine-vision synchronization (MVS) software solution designed to address the pain points inherent to TEM studies. Once installed on the microscope, it enables the continuous synchronization of images and metadata generated by the microscope, detector, and in situ systems during an experiment. This connectivity enables the application of machine-vision algorithms that apply a combination of spatial, beam, and digital corrections to center and track a region of interest within the field of view and provide immediate image stabilization. In addition to the substantial improvement in resolution afforded by such stabilization, metadata synchronization enables the application of computational and image analysis algorithms that calculate&#8239;variables between images. This calculated metadata can be used to analyze trends or identify key areas of interest within a dataset, leading to new insights and the development of more sophisticated machine-vision capabilities in the future. One such module that builds on this calculated metadata is dose calibration and management. The dose module provides state-of-the-art calibration, tracking, and management of both the electron fluence (e-/&#197;2&#183;s-1) and cumulative dose (e-/&#197;2) that is delivered to specific areas of the sample on a pixel-by-pixel basis. This enables a comprehensive overview of the interaction between the electron beam and the sample. Experiment analysis is streamlined through a dedicated analysis software in which datasets consisting of images and corresponding metadata are easily visualized, sorted, filtered, and exported. Combined, these tools facilitate efficient collaborations and experimental analysis, encourage data mining and enhance the microscopy experience.

تسليط الضوء على المؤلف: نهج رؤية الآلة لسير عمل المجهر الإلكتروني النافذ وتحليل النتائج وإدارة البيانات  

Here, we present a protocol to utilize machine-vision software to stabilize dynamic processes during TEM imaging, while concurrently indexing multiple streams of metadata to each image into a navigable timeline. We demonstrate how this platform enables automated calibration and mapping of the electron dose over the course of an experiment.