תודה מיוחדת קריס Reddi ב-UCLA להגדרת ריאגנטים לשפוך ג&#39;ל וכדי ארין סנדרס באוניברסיטת קליפורניה השראה, הדרכה, תמיכה והגהה את כתב היד. אני רוצה גם תודה ג&#39;אנקרלו Costaguta וגרגורי ס פיין לאספקת הגנומי שמרים דנ&quot;א primers כדי להגביר את הגן GAL3. אני רוצה גם להודות שאה Bhairav ​​לצילום תמונות של ציוד מעבדה חומרים כימיים המשמשים לייצור דמויות 2 - 4. המימון לפרויקט זה סופק על ידי HHMI (HHMI גרנט מס &#39;52006944).

<ol>
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אין לי מה לחשוף.

PCR הפך לכלי הכרחית בארסנל מדע הביולוגיה. PCR שינתה את מהלך המדע המאפשר ביולוגים להניב כוח על הגנום, ולהפוך גנים היברידית עם פונקציות חדשות, המאפשר ניסויים קליניים ספציפי ומדויק, צובר תובנות לתוך הגנום לבין גיוון, כמו גם שיבוט גנים פשוט לניתוח ביוכימי נוסף. יישום ה-PCR מוגבל רק על ידי הדמיון של המדען אשר מפעיל את כוחו. יש הרבה ספרים וניירות מיוחדים המתארים את השימושים החדשים של ה-PCR, ועוד רבים יפותח על הדור הבא של מדע הביולוגיה. עם זאת, ללא קשר הגישות הצפויות, את המסגרת הבסיסית נשארה זהה. PCR, בתוך כל הפאר שלה, הוא יישום במבחנה כדי לייצר כמויות גדולות של קטע מסוים של דנ&quot;א. תכנון הניסוי PCR דורש מחשבה וסבלנות. התוצאות המוצגות באיור 3 מדגימים אחד פרק מרכזיallenges בעת תכנון אסטרטגיית אופטימיזציה של ה-PCR. כלומר, כאשר פרמטר אחד של PCR משתנה, זה יכול להשפיע אחרת. לדוגמה, אם ה-PCR הראשוני בוצע בטמפרטורה תת אופטימלית חישול (58 ° C) עם Mg 2 + ריכוז אופטימלי של mM 2.0, אז התוצאה היתה לייצר כתם כפי שניתן לראות באיור 3 ג. ניסיון לפתור את משטח עשוי לכלול הגדרת תנאי ה-PCR עם תגובות המכילות 2.0 mM MgCl 2 והתאמת הטמפרטורה חישול 61 ° C. עם זאת, כפי שניתן לראות באיור 3 א, זו לא תניב כל מוצר. כתוצאה מכך, רצוי לכיל ריאגנטים, במקום להוסיף 1 ריכוז לתגובה אחת, כאשר פתרון בעיות תוצאות מזויפות. כמו כן, את ההתאמות הנפוצות ביותר הדרושות לייעול ניסוי ה-PCR הם לשנות את ריכוז 2 מ&quot;ג + ולתקן הטמפרטורות חישול. עם זאת, אם שינויים אלה אינם לצמצם או לבטל תוצאות חריגים, טיטרציה של additiVes ו / או לשנות את מצבו אופניים פרוטוקולים כמתואר טבלאות 2-6 עשוי להקל על הבעיה. אם כל השאר נכשל, לעצב מחדש את primers ונסה, ונסה שוב.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> Taq Polymeras </td><td> סיגמא </td><td> D4545-25UN </td><td></td></tr><tr><td> dNTPs </td><td> Qiagen </td><td> 201225 </td><td></td></tr><tr><td> 0.2 מ&quot;ל דק וול צינורות צינורות PCR </td><td> ביו רד </td><td> 223-9469 </td><td></td></tr><tr><td> 0.2 מ&quot;ל דקים קיר כמוסות תחתית </td><td> ביו רד </td><td> 223-9472 </td><td></td></tr><tr><td> TE חוצץ: </td><td></td><td></td><td></td></tr><tr><td> EDTA הרכב: dihydrate מלח </td><td> סיגמא </td><td> E5134 </td><td></td></tr><tr><td> Trizma-HCl </td><td> סיגמא </td><td> T-3253 </td><td></td></tr><tr><td> הפאג מותאם אישית פריימר </td><td> Invitrogen </td><td colspan="2"> 25441F_RL6_Contig2_68k TACTGCAACGCGATGTTGCG </td></tr><tr><td> הפאג מותאם אישית פריימר </td><td> Invitrogen </td><td colspan="2"> 26007R_RL6_ Contig2_68k TCACCATCAATCGTGGCGGT </td></tr><tr><td> שמרים מותאם אישית פריימר </td><td> Integrated DNA Technologies </td><td colspan="2"> SacI-Gal3 (-256) ACCTGGAGCTCCTATTT TAACATGTGGATTTCTTGAAAGAATGAAATCG </td></tr><tr><td> שמרים מותאם אישית פריימר </td><td> Integrated DNA Technologies </td><td colspan="2"> Gal3 (1848), SacI CGGATGGAGCTCGCGT GAAGGTAATTTTTTTTTATGTCTCCG </td></tr><tr><td> תרמית Cycler </td><td> ביו רד </td><td> 170-9703 </td><td> שלי Cycler </td></tr><tr><td> Agarose </td><td> ISC BioExpress </td><td> E-3120-500 </td><td></td></tr><tr><td> ג&#39;ל אלקטרופורזה </td><td> Hoeffer מכשירים מדעיים </td><td> דגם HE33 </td><td></td></tr><tr><td> 1 קילו סולם DNA </td><td> ניו אינגלנד Biolabs </td><td> N3232S </td><td></td></tr><tr><td> ביו רד Power Pack </td><td> ביו רד </td><td> 164-5050 </td><td> PowerPac בסיסי </td></tr><tr><td> ג&#39;ל דוק מערכת </td><td> Fotodyne Incorporated </td><td colspan="2"> Foto / אנליסט חוקר תחנת עבודה FX </td></tr></tbody></table>

polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies

במדעים הביולוגיים היו ההתקדמות הטכנולוגית, כי מעוט משמעת לתוך גיל הזהב של גילוי. לדוגמה, בתחום המיקרוביולוגיה הפך עם כניסתו של המיקרוסקופ אנטון ואן לוונהוק, שאיפשרה למדענים לחזות פרוקריוטים בפעם הראשונה. התפתחות התגובה פולימראז שרשרת (PCR) הוא אחד מאותם חידושים ששינו את מהלך המדע מולקולרית עם השפעתה פורש subdisciplines אינספור בביולוגיה. תהליך תיאורטי קווי המתאר של Keppe ועמיתים לעבודה בשנת 1971, עם זאת, זה היה עוד 14 שנים עד הליך שלם PCR תואר ויישם באופן ניסיוני על ידי Kary מוליס תוך Cetus החברה בשנת 1985. אוטומציה ושכלול של טכניקה זו התקדם עם הקדמה של פולימראז ה-DNA תרמית יציבה מ aquaticus החיידק Thermus, וכתוצאה מכך פולימראז שם Taq DNA. PCR הוא poweהגברה rful טכניקה שיכולה ליצור היצע נרחב של קטע מסוים של דנ&quot;א (כלומר, amplicon) מתוך כמות קטנה בלבד של חומר המוצא (כלומר, תבנית ה-DNA או רצף המטרה). אמנם פשוט, ובדרך כלל ללא בעיות, יש מלכודות שיסבכו את התגובה לייצר תוצאות מזויפות. כאשר ה-PCR נכשל זה יכול להוביל שאינם ספציפיים מוצרים רבים דנ&quot;א בגדלים שונים המופיעים כסולם או כתם של להקות על ג&#39;ל agarose. לפעמים מוצרי שום צורה כלל. בעיה נוספת מתרחשת כאשר הפוטנציאל מוטציות פוגש בטעות ב amplicons, וכתוצאה מכך לאוכלוסייה הטרוגנית של מוצרי ה-PCR. כשלים PCR יכול להיות מתסכל, אלא אם כן סבלנות פתרון בעיות זהיר מועסקים לברר ולפתור את הבעיה (S). פרוטוקול זה מתאר את העקרונות הבסיסיים של ה-PCR, מספק מתודולוגיה יביא הגברה של רצפי היעד ביותר, מציג אסטרטגיות למיטוב התגובה. על ידי ביצוע מדריך זה PCR, רח&#39;udents צריכים להיות מסוגלים: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדר את התגובות ותנאי רכיבה תרמית לניסוי ה-PCR קונבנציונאלי </li><li style=";text-align:right;direction:rtl"> להבין את תפקידם של מרכיבי התגובה שונים ואת השפעתם הכוללת על ניסוי ה-PCR </li><li style=";text-align:right;direction:rtl"> עיצוב לייעל הניסוי PCR עבור כל תבנית ה-DNA </li><li style=";text-align:right;direction:rtl"> פתרון ה-PCR ניסויים כושלים </li></ul>

Watch this Scientific Journal Video about Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies at JoVE.com

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

PCR התפתחה טכניקה נפוצה הרבה מעבדות ביולוגיה מולקולרית. בתנאי הנה מדריך מהיר קונבנציונאלי פרוטוקולים מספר ה-PCR. כי כל תגובה היא ניסוי ייחודי, תנאים אופטימליים הנדרשים כדי לייצר מוצר להשתנות. הבנת המשתנים התגובה יהיה מאוד לשפר את יעילות לפתרון בעיות, ובכך להגדיל את הסיכוי להשיג את התוצאה הרצויה.

תגובת שרשרת פולימרז: פרוטוקול בסיסי פלוס אסטרטגיות לפתרון בעיות ואופטימיזציה

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

polymerase-chain-reaction-basic-protocol-plus-troubleshooting

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399.7K Views.  University of California, Los Angeles. PCR התפתחה טכניקה נפוצה הרבה מעבדות ביולוגיה מולקולרית. בתנאי הנה מדריך מהיר קונבנציונאלי פרוטוקולים מספר ה-PCR. כי כל תגובה היא ניסוי ייחודי, תנאים אופטימליים הנדרשים כדי לייצר מוצר להשתנות. הבנת המשתנים התגובה יהיה מאוד לשפר את יעילות לפתרון בעיות, ובכך להגדיל א?...

Video: Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

אופטימיזציה של התגובה אוגי באמצעות סינתזה במקביל וטיפול נוזלי אוטומטי

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

מבוך פלוס גבוהות עבור עכברים

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

פולשנית In vivo קטנים בעלי חיים MRI ו MRS: הפרוצדורות בסיסי

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

מתודולוגיה multiplexed Fluorometric immunoassay בדיקה ופתרון בעיות

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

בסיסי<em&gt; Caenorhabditis elegans</em&gt; שיטות: סנכרון תצפית

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

מדריך למודרני כמותי פלורסנט המערבי סופג עם אסטרטגיות לפתרון בעיות

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

אסטרטגיה כדי לאמת את התפקיד של Gating Plasmodesmal בתיווך Callose במענה חוג

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

פרוטוקול אוטומאטי ומהיר עבור שריר סיבי ניתוח אוכלוסיית חתכי שריר עכברוש באמצעות שרשרת השרירן כבד אימונוהיסטוכימיה

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

הליך ואסטרטגיות אופטימיזציה מפתח שיטה אוטומטית נימי מתכלה מבוסס על Electrophoretic

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

מדידות סימולטני של סידן תאיים, פוטנציאל ממברנה אנדותל מוחי העכבר טרי מבודדת ללא פגע

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...