חשוב מאוד ללבוש אבקת ללא כפפות במהלך תקופת ההכנה של שכבת דק. ניקוי הצלחת מדגם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש נירוסטה או ציפוי זהב MALDI המדגם. </li><li style=";text-align:right;direction:rtl"> לשטוף עם MeOH ולנגב בעדינות עם Kimwipe. אל תשפשפו או תגרדו את פני השטח עם Kimwipe, כדי למנוע מגרד את פני השטח. </li><li style=";text-align:right;direction:rtl"> לשטוף עם H 2 O ולנגב בעדינות עם Kimwipe. </li><li style=";text-align:right;direction:rtl"> לשטוף עם MeOH ולנגב בעדינות עם Kimwipe. </li><li style=";text-align:right;direction:rtl"> אם יש צורך, חזור MeOH / H 2 O / MeOH מחזור ניקוי, תמיד וכלה MeOH. </li><li style=";text-align:right;direction:rtl"> ודא כי אין כתמים רפאים או שאריות שנותרו על הצלחת. אם יש כתמים רפאים, לשטוף את הצלחת במים deionized תוך שפשוף פני השטח עם יד בכפפה שלך (לא לשפשף עם Kimwipes). חזור על MeOH / H 2 O / MeOH מחזור הניקוי. </li><li style=";text-align:right;direction:rtl"> השתמש צלחת מיד לאחר הכביסה. </li></ol> הכנת הפתרון שכבה דקה המצע <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לערבב 1 חלק רווי 4-HCCA (2 חלקים ACN, 1 חלק מים, ו - 0.1% TFA סופי) ו 3 חלקים isopropanol. הערה: פתרון דק שכבת המצע הוא יציב מאוד, אפשר לשמור בטמפרטורת החדר בחושך במשך שנה. זה עשוי גם להיות מאוחסן ב -4 ° C. </li></ol> ביצוע המצע שכבה דקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> החל 20-50μL של הפתרון המצע שכבה דקה על מרכז שמאל של הצלחת, ופרש עם הצד של הטיפ פיפטה. טאטא בכיוון אחד. </li><li style=";text-align:right;direction:rtl"> ככל מתייבש פתרון, מתאדה ממס אורגני מהר מאוד, והותיר אחריו טיפות זעירות של מים על פני הצלחת. בשלב זה, לעטוף את האצבע המורה עם רובדי של Kimwipe ובתחילה כתם בעדינות את פני השטח של צלחת עם זה. </li><li style=";text-align:right;direction:rtl"> כאשר פני השטח הוא משתחרר מן טיפות מים, לנגב את הצלחת עם כל חד כיוונית תנועות רחבות באמצעות Kimwipe. הערה: אם הפתרון אינו מספיק להתפשט בכל רחבי השטח צלחת, להגדיל את הרכב ACN ו מחדש את שכבה דקה. כמה פרמטרים שעשויים להשפיע על התפשטות של הפתרון כוללים חום ולחות הסביבה. הערה: עם צלחות זהב, שכבת מופיע בדרך כלל כהשתקפות צהבהב, תלוי זוויות צפייה אור. עם לוחות פלדה, רק את הקצה של השכבה גלוי בדרך כלל. </li><li style=";text-align:right;direction:rtl"> נקו את הקצוות של צלחת עם Kimwipe טרי לפני הצבת אותו מחזיק את הצלחת כדי למנוע זיהום של מטריקס מכשיר. </li></ol> הכנת התמיסה מטריקס <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> <a href="http://prowl.rockefeller.edu/protocols/saturated-4HCCA-solution.html">רווי 4-HCCA</a> ב FWI (3 חלקים חומצה פורמית, 1 חלק מים, 2 חלקים isopropanol) מומלץ ניתוח קרום והן חלבונים מסיסים. זה יכול לשמש גם במקרים מיוחדים לצורך ניתוח של פפטידים גדולים מאוד הידרופובי (M&gt; 4000 u). ודא כי פתרון מטריצה ​​המשמשת תצפית החלבונים אינו עולה ~ תוכן אורגניים 50%, כפי שהוא לחלוטין לפזר את המצע שכבה דקה, להביס את היתרונות של השיטה. </li></ol> מבחן של המצע שכבה דקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ספוט 0.5μL מהפתרון מטריקס על הצלחת. משקע לבנבן יופיעו בממשק שבין שכבת דק ואת רביב. כאשר שכבה זו מכסה את החלק התחתון של אגל לחלוטין, בדרך כלל בתוך 10-15 שניות, לשאוב את הנוזל העודף עם קו ריק. אם זה לוקח יותר זמן מאשר 30 שניות לזרז את הטופס, הוא אינדיקציה לכך שאולי יש בעיה עם המצע שכבה דקה ו / או פתרון מטריקס. </li></ol> לדוגמה יישום <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ריכוז אנליטי של פתרונות המוצא צריכה להיות בטווח של 10 ו 1000μM. </li><li style=";text-align:right;direction:rtl"> לדלל את פתרון אנליטי בפתרון מטריצה ​​כדי ריכוז אנליטי הסופי בין 0.2 ל 2μM. למשל, להתחיל עם 1μl של מדגם 9μl של פתרון מטריקס. הערה: ודא כי המטריצה ​​קרוב להיות רווי הפתרון הסופי, אחרת אתה תהיה redissolve המצע שכבה דקה. הערה: ריכוזים אנליטי נדרש לקבל אותות הגון הם תלויים במידה רבה על מורכבותו פתרון הרכב (למשל, מזהמים) ו ionizability אנליטי. הערה: אם דילול בשלב יחיד אינו להניב תוצאות טובות, כדאי לך לנסות עוד צעד דילול. למרות נגדית אינטואיטיבי, פתרונות אנליטי מרוכז מאוד נדירות לייצר ספקטרום טוב. הערה: חלבונים ממברנה בדרך כלל דורשים דילולים גדולים יותר. </li><li style=";text-align:right;direction:rtl"> ספוט 0.5μl תערובת מדגם / מטריצה ​​על הצלחת. משקע לבנבן יהוו בממשק שבין שכבת דק ואת רביב. משקע זה cocrystallization של מטריקס אנליטי. כאשר שכבה זו מכסה את החלק התחתון של אגל לחלוטין, usuברית בתוך 10-15 שניות, לשאוב את הנוזל העודף עם קו ריק. אם זה לוקח יותר מ -30 שניות עבור לזרז את הטופס, זה עשוי להיות אינדיקציה להציג המזהם, אשר מונע התגבשות, או אנליטי שלך מרוכז מדי. </li></ol> Calibrant יישום <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מכינים את תערובת calibrant / מטריצה ​​באופן דומה עם ריכוז calibrant הסופי בין 0.1-0.5μM. </li><li style=";text-align:right;direction:rtl"> ספוט 0.5μl calibrant / מטריצה ​​התערובת על הצלחת בסמיכות הנקודות המדגם. קרבה פיזית זו משמש הליך מדומה כיול פנימי שבו צלחת מועבר מן המקום מדגם למקום calibrant במהלך הרכישה המדגם, כדי להוסיף כמה יריות לייזר calibrants כדי הספקטרום. גישה זו מבטיחה דיוק גבוהה יותר מאשר המסה כיול כיול חיצוני בלבד. </li></ol> כביסה צעד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשטוף עם כל נקודה כ 2μL של פתרון קרים כקרח 0.1% TFA מימית. לשאוב נוזל עודף עם קו ריק. </li></ol> רכישת ספקטרום המונית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כעת אתה מוכן לרכוש ספקטרה המונית. אם אתה משתמש בפתרון מטריצה ​​FWI, עליך לרכוש ספקטרה שלך בהקדם האפשרי כדי למנוע שינוי בלתי הפיך אנליטי, כגון formylation. </li></ol>

<ol>
	<li>Xiang, F., Beavis, R. C. <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+to+Increase+Contaminant+Tolerance+in+Protein+Matrix-Assisted+Laser+Desorption+Ionization+by+the+Fabrication+of+Thin+Protein-Doped+Polycrystalline+Films.">A Method to Increase Contaminant Tolerance in Protein Matrix-Assisted Laser Desorption Ionization by the Fabrication of Thin Protein-Doped Polycrystalline Films.</a> Rapid Communications in Mass Spectrometry. 8, 199-204 (1994).</li><li>Cadene, M., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Robust,+Detergent-Friendly+Method+for+Mass+Spectrometric+Analysis+of+Integral+Membrane+Proteins.">A Robust, Detergent-Friendly Method for Mass Spectrometric Analysis of Integral Membrane Proteins.</a> Analytical Chemistry. 72, 5655-5658 (2000).</li><li>Hook, P., Mikami, A., Shafer, B., Chait, B. T., Rosenfeld, S. S., Vallee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+allosteric+control+of+cytoplasmic+dynein+ATPase+activity+by+the+stalk+and+C-terminal+domains.">Long-range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains.</a> J Biol Chem. 280, 33045-33054 (2005).</li></ol>

זה קריטי, כי כל ריאגנטים הם באיכות הגבוהה ביותר (כגון HPLC כיתה). אבקת ללא כפפות (לטקס או nitrile) נירוסטה או ציפוי זהב MALDI מדגם מתנול (MeOH) מים Kimwipes אלפא cyano-4-hydroxycinnamic חומצה (4-HCCA) Trifluoroacetic חומצה (TFA) (הריכוז הסופי של 0.1% ב H 2 O) אצטוניטריל (ACN) חומצה פורמית Isopropanol

maldi sample preparation: the ultra thin layer method

וידאו זה מדגים את הכנת שכבת מטריצה ​​/ אנליטי דקים לניתוח פפטידים וחלבונים על ידי מטריקס בסיוע לייזר desorption Ionization המוניים ספקטרומטריית (MALDI-MS) 1, 2. השיטה דקים שכבת כולל ייצור של שכבת המצע של גבישים מטריקס (אלפא cyano-4-hydroxycinnamic חומצה) על הצלחת המדגם, אשר משמש קרקע זריעת לגיבוש הבאות של תערובת מטריצה ​​/ אנליטי. יתרונות השיטה דקים שכבת פני גישות אחרות בתצהיר מדגם (טיפה מיובשים למשל) הם שהוא מספק (i) סובלנות גדולה יותר זיהומים כמו מלחים דטרגנטים, (ii) רזולוציה טובה יותר, וכן (iii) אחידות מרחבית גבוהה יותר. שיטה זו שימושית במיוחד לקביעת המסה המדויקת של החלבונים. פרוטוקול פותחה בתחילה אופטימיזציה עבור ניתוח של חלבונים בממברנה ומשמש בהצלחה לנתח תעלות יונים, מובילי המטבוליט, ואת הקולטנים, המכיל בין 2 ל 12 תחומים הטרנסממברני 2. מאז פרסום מקורי, הוא הראה גם להיות שימושי באותה מידה לניתוח חלבונים מסיסים. אכן, יש לנו השתמשתי בה עבור מספר גדול של חלבונים בעל מגוון רחב של תכונות, כולל אלה עם ההמונים מולקולרי גבוה ככל 380 kDa 3. עכשיו שיטת הבחירה שלנו לצורך ניתוח המסה המולקולרית של כל החלבונים. ההליך המתואר באופן עקבי מייצרת באיכות גבוהה ספקטרה, והוא רגיש, חזק, וקל ליישום.

Watch this Scientific Journal Video about MALDI Sample Preparation: the Ultra Thin Layer Method at JoVE.com

MALDI Sample Preparation: the Ultra Thin Layer Method

וידאו זה מדגים את הכנת שכבת מטריצה ​​/ אנליטי דקים לניתוח פפטידים וחלבונים על ידי מטריקס בסיוע לייזר desorption Ionization המוניים ספקטרומטריית (MALDI-MS).

MALDI הכנה לדוגמא: שיטת אולטרה שכבה דקה

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

maldi-sample-preparation-the-ultra-thin-layer-method

Research

JoVE Journal

Biology

19.2K Views.  Rockefeller University. וידאו זה מדגים את הכנת שכבת מטריצה ​​/ אנליטי דקים לניתוח פפטידים וחלבונים על ידי מטריקס בסיוע לייזר desorption Ionization המוניים ספקטרומטריית (MALDI-MS).

Video: MALDI Sample Preparation: the Ultra Thin Layer Method

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

המערבי סופג: הכנת המדגם איתור

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Thaliana ארבידופסיס פולאר Glycerolipid פרופיל ידי כרומטוגרפיה בשכבה דקה (TLC) יחד עם כרומטוגרפיה גז נוזלי (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

הכנת למבוגרים תסיסנית עיניים עבור חתך דק ניתוח מיקרוסקופי

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

לדוגמא הכנה לניתוח Cytometry Mass

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

הכנת הדוגמא והדימות של Exosomes על ידי במיקרוסקופ אלקטרונים הילוכים

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

הכנת הדוגמא ולמחקר במיקרוסקופ אלקטרונים הילוכים

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

הפיוניות הקלטת-פיל: שיטה משופרת עבור הכנת הדוגמא משמר תא

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

הכנת הדוגמא מסה ספקטרומטר מבוססת על ניתוח פרוטאומיקס של עינית Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

הכנת הדוגמא ביולוגית על ידי הקפאת בלחץ גבוה, שיפור ניגודיות בסיוע מיקרוגל, ושרף מינימלי הטמעה של אמצעי הדמיה

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

באמצעות Microtiter צלחת Radiolabeling עבור מספר מדידות Vivo של es, coli (p) ppGpp ואחריו כרומטוגרפיה שכבה דקה

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...