עבודה זו נתמכה על ידי ארצות הברית בפרס אישי שירות הבריאות NS-071530.

1. הכנת תאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדול יותרת המוח AtT20 תאים במדיום שונה Dulbecco של הנשרים (DMEM) השלימו עם סרום סוס 10% ולשמור על תרבויות על 37 מעלות צלזיוס באווירה humidified של 5% CO 2. </li><li style=";text-align:right;direction:rtl"> לשמור על התרבות התאים על ידי subculturing כל 5 עד 7 ימים באמצעות הליך trypsinization סטנדרטי. </li><li style=";text-align:right;direction:rtl"> לצפות את הבארות של התחתון, שחור בהיר, צלחות 96-50 גם עם μL של פולי-L-ליזין. אפשר בארות את לייבוש למשך 30 דקות בחממה. </li><li style=";text-align:right;direction:rtl"> הצלחת התאים את הצלחות 96-200 המכילים גם התקשורת μL בצפיפות של 30,000 תאים בכל טוב לאחסן את התאים בחממה של 3-4 ימים. </li><li style=";text-align:right;direction:rtl"> השתמש ההתקנה השאיפה, בהיקף של צנצנת 4 ליטר פקוק מחובר ואקום בצד אחד טיפ 200 פיפטה μL בקצה השני, לאט לאט לשאוב בינוני תרבות monolayer מן התא. </li><li style=";text-align:right;direction:rtl"> שוטפים את התאים עם μL 200 של פתרון חיץ רגיל (132 mM NaCl, KCl 5 מ&quot;מ, 1 מ &#39;M CaCl 2, 1 mM MgCl 2, 5 מ&quot;מ דקסטרוז, 5 HEPES מ&quot;מ, ה-pH 7.4 עם NaOH). </li><li style=";text-align:right;direction:rtl"> הוספת 200 μL של פתרון מאגר המכיל את הפוטנציאל רגיש קרום צבע (MPD) DiBAC 4 (3) או HLB 021-152 (5 מיקרומטר) זה טוב דגירה התאים דקות 40 ב 37 ° C. </li></ol> 2. הכנת תרכובות בדיקה וטיפול Cell <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן דילול סדרתי של תרכובות הבדיקה (מניות = 10 מ&quot;מ DMSO) מספריית מתחם (בפורמט 96-היטב) באמצעות (ThermoFisher) Versette טיפול אוטומטי מערכת נוזלי. </li><li style=";text-align:right;direction:rtl"> הסר את צלחת 96-ובכן, המכיל את AtT20 תאים, מן החממה ולאפשר הצלחת לחזור לטמפרטורת החדר. לשאוב את המאגר הישן וליישם פתרון חוצץ חדש המכיל את MPD אל התאים. </li><li style=";text-align:right;direction:rtl"> באמצעות מערכת טיפול הנוזל להחיל ריכוזים שונים (10 ננומטר ל -10 מיקרומטר) של תרכובות הבדיקה (2-20 μL) ופתרונות שליטה (0.1% DMSO) (במאגר פתרון containing MPD) לצלחת 96-המכילה גם את התאים. </li><li style=";text-align:right;direction:rtl"> דגירה התאים דקות 5 עם תרכובות הבדיקה שקדמו המדידות ניאון. </li></ol> 3. הפעלת ערוצים GIRK, מדידה וניתוח פלורסנט ו <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנס את צלחת 96-היטב לקורא Biotek צלחת Synergy2 ניאון ולקבל את האות רקע פלורסנט באורכי גל עירור ופליטה של ​​520 ו 560 ננומטר, בהתאמה. </li><li style=";text-align:right;direction:rtl"> החל את ligands GPCR (= סומטוסטטין 200 מייל ימי או carbachol = 10 מיקרומטר) או פתרון שליטה (מאגר לבד) (20 μL) אל בארות (הנפח הכולל = 220 μL) להפעלת ערוצי GIRK באמצעות מערכת ההזרקה Synergy2. איסוף נקודות נתונים במרווחי זמן של 10 פני תקופה דגימה של 300 באורכי גל עירור ופליטה של ​​520 ו 560 ננומטר, בהתאמה. </li><li style=";text-align:right;direction:rtl"> קבל את הקורס בזמן שיא האמפליטודה של שינוי ניאון באמצעות Biotek Gen5 תוכנה לייצא את הנתונים ל-Excel לצורך ניתוח נוסף. </lאני&gt; <li style=";text-align:right;direction:rtl"> לחשב את Z&#39;גורמים כדי לקבוע את המהימנות של assay. לוחות המשמשים לקביעת Z&#39;גורם מכיל 2 שורות כל אחד חיובי (ליגנד GPCR) והשלילי (מאגר לבד) שולטת. Z&#39;גורם מוגדר: Z &#39;= 1 - (P + 3σ 3σ N) / | P μ - μ N |, כאשר μ μ P &amp; N הן אמצעי שליטה חיובית אותות בקרה שליליות, ו σ P &amp; σ N הן סטיות תקן של שליטה חיובית אותות בקרה שליליות, בהתאמה 6. Z&#39;גורמי בטווח של 0.5-1.0 עולה כי איכות assay מצוין 6. צלחות עם גורמי Z&#39;מתחת 0.5 אינם נכללים בניתוח. </li><li style=";text-align:right;direction:rtl"> תרכובות לווסת את האות ניאון הם נוספת שתבצע בנוכחות Ba 2 + או tertiapin-Q כדי לאשר פנימה מיישר K + הספציפיות הערוץ. </li></ol> 4. Representative תוצאות למשל האות הקרינה נמדדת באמצעות assay ערוץ GIRK מוצג באיור 2. תוספת של somatostatin ליגנד GPCR (200 ננומטר) ל AtT20 תאים הנגרם מהיר, הזמן תלוי ירידה האות HLB 021-152 ניאון (איור 2). לעומת זאת, התוספת של פתרון בקרת המיוצר עלייה מיידית קטנה הקרינה שעם הזמן חזר לקו הבסיס (איור 2). השוואה בין ערכים ניאון השיא ב 96-הזריק גם צלחות עם שליטה ופתרונות somatostatin נתן כמה Z&#39;גורמי בטווח של 0.5-0.7. עבור כימות נוספת, הרשומה השליטה היה מפחיתים את שיא somatostatin ואת התוצאה somatostatin רגיש האות ניתח (איור 2). Assay מספק שיטה מהירה לזיהוי תרופות לווסת ערוצי GIRK. לדוגמה, תרופות המעכבות את צ&#39;ה GIRKnnel צריך להפחית GPCR ליגנד בתיווך ירידה הקרינה על ידי מניעת K + בזרימת מן התאים. כפי שניתן לראות באיור 3, טיפול של התאים עם tertiapin-Q, רעלן זה חוסם ערוצי GIRK 7, המיוצר על עיכוב של שינוי somatostatin בתיווך ניאון. Propafenone, תרופה אנטי בקצב לא סדיר שחוסמת ערוצי GIRK בלב 8, גם עכבות שינוי ניאון (איור 4). Assay יכול גם להיות שימושי עבור זיהוי activators של ערוץ GIRK. עם זאת, היישום של אתנול (100 &amp; 200 מ&quot;מ), גרמה activator של ערוץ GIRK 2,3, לא חל שינוי משמעותי אות ניאון בהשוואה לשלוט פתרון (p&gt; 0.5). <img src="/files/ftp_upload/3850/3850fig1.jpg" alt="איור 1"/> באיור 1. עיצוב ניסיוני של assay GIRK ערוץ ניאון. AtT20 תאים מודגרת במאגר המכיל membranדואר פוטנציאל רגיש צבע פלואורסצנטי (D). את מולקולות צבע להיכנס לתאים ולהפוך ניאון על קשירה חלבונים תאיים (פאנל למעלה). עקידת somatostatin (סום) כדי GPCR שלה מגרה את החלבון מדכא G (G i) גורם להפעלה של ערוץ GIRK (הפאנל התחתון). בזרימת הבאות של K + מחוץ לתאים גורמת קרום הפוטנציאל להיות שלילי יותר את מולקולות צבע כדי לצאת התאים. כתוצאה מכך את האות יורדת ניאון (הפאנל התחתון). <img src="/files/ftp_upload/3850/3850fig2.jpg" alt="איור 2"/> 2. איור נציג HLB 021-152 אות ניאון נמדד לאורך זמן את AtT20 תאים במהלך תוספת של somatostatin או פתרון שליטה. היחס בין עוצמת ניאון (F / F O) חושב על ידי חלוקת האות בנוכחות (ו) somatostatin (או פתרון שליטה) על ידי האות המחקר נמדד לפני (F O) מודעותdition של somatostatin (או פתרון שליטה). כל נקודה מייצגת ממוצע ± SE שהושגו 5-6 בארות. Somatostatin או פתרון שליטה נוסף בזמן אפס (↓). התוספת של פתרון בקרת גרם לעלייה חולפת קטן אות ניאון. זו תוצאה של שינוי הטמפרטורה הנגרמת על ידי הזרקה של הפתרון. <img src="/files/ftp_upload/3850/3850fig3.jpg" alt="איור 3"/> איור 3. טיפול של AtT20 תאים עם tertiapin-Q (500 ננומטר) מעכב את הירידה somatostatin בתיווך האות ניאון באמצעות קשירה וחסימת ערוצי GIRK. כל נקודה מייצגת ממוצע ± SE שהושגו 4-6 בארות. Somatostatin נוספה בזמן אפס (↓). <img src="/files/ftp_upload/3850/3850fig4.jpg" alt="איור 4"/> איור 4. טיפול של AtT20 תאים עם propafenone (20 מיקרומטר) גם מעכב ירידה somatostatin בתיווך באות ניאון. כל נקודה מייצגת ממוצע ± SE שהושגו 5-6 בארות. Somatostatin נוספה בזמן אפס (↓).

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למרות הפוטנציאל רגישים קרום צבעי ניאון נעשה שימוש כדי לזהות תרופות אשר לווסת תעלות יונים 9,10, זהו הדו&quot;ח הראשון של בקשתם העצבית GIRK ערוץ גילוי תרופות. Assay ערוץ GIRK ניאון המובא כאן מספק שיטה מהירה, אמינה בזמן אמת להקרנה של ליגנד, מגודרת K + ערוצים. Assay ניתן לשנו?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> DMEM </td><td> CellGro </td><td> 10-013 </td></tr><tr><td> סוס סרום </td><td> Invitrogen </td><td> 16050-114 </td></tr><tr><td> 96-גם צלחות </td><td> קורנינג </td><td> 3603 </td></tr><tr><td> פולי-L-ליזין </td><td> Sigma-Aldrich </td><td> P4707 </td></tr><tr><td> Somatostatin </td><td> Sigma-Aldrich </td><td> S9129 </td></tr><tr><td> Carbachol </td><td> Sigma-Aldrich </td><td> C4382 </td></tr><tr><td> HLB 021-152 </td><td> AnaSpec </td><td> 89300 </td></tr><tr><td> Versette המטפל נוזל אוטומטי </td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> Synergy2 צלחת ניאון הקורא </td><td> Biotek </td><td></td></tr><tr><td> Gen5 ניתוח שלoftware </td><td> Biotek </td><td></td></tr></tbody></table> טבלה 1. טבלה של חומרים כימיים מסוימים וציוד.

a fluorescent screening assay for identifying modulators of girk channels

G-חלבון סגורות פנימה מיישר K + (GIRK) ערוצים לתפקד כמתווכים הסלולר של מגוון רחב של הורמונים נוירוטרנסמיטורים באים לידי ביטוי השרירים, המוח, הלב ורקמות שלד 1,2 האנדוקרינית. ערוצי GIRK להיות מופעל בעקבות עקידת ligands (נוירוטרנסמיטורים, הורמונים, תרופות וכו &#39;) כדי פלזמה שלהם קרום הנכנס, G חלבונים מצמידים קולטנים (GPCRs). מחייב זה גורם לגירוי של חלבונים G (G I ו-G o) אשר לאחר מכן לאגד ולהפעיל את ערוץ GIRK. פעם אחת פתחתי את ערוץ GIRK מאפשר תנועה של K + מחוץ לתא גורמת קרום מנוחה הפוטנציאל להיות שלילי יותר. כתוצאה מכך, הפעלת GIRK ערוץ בנוירונים פוחתת היווצרות ספונטנית פוטנציאל פעולה מעכב שחרור של שליחים עצביים מעוררים. בלב, הפעלת ערוץ GIRK מעכב פעילות הקוצב ובכך להאט את קצב הלב. <p= &quot;jove_content&quot; הכיתה&gt; ערוצי GIRK לייצג מטרות חדשות לפיתוח תרופות טיפוליות חדשות לטיפול בכאב נוירופתי, התמכרות לסמים, הפרעות בקצב הלב והפרעות אחרות 3. עם זאת, פרמקולוגיה של ערוצים אלה עדיין נודעת בעיקר. למרות מספר תרופות כולל נגד סוכני בקצב לא סדיר, תרופות אנטי פסיכוטיות וכן תרופות נוגדות דיכאון לחסום את הערוץ GIRK, עיכוב זה אינו סלקטיבי מתרחש בריכוזים גבוהים יחסית סמים 3. כאן אנו מתארים assay בזמן אמת ההקרנה לזיהוי מאפננים חדשים של ערוצי GIRK. ב assay זה, תאים עצביים AtT20, אשר הביעו את ערוצי GIRK, נטענים עם פוטנציאל רגיש צבעים קרום ניאון כגון bis-(1,3-dibutylbarbituric חומצה) trimethine oxonol [DiBAC 4 (3)] או HLB 021-152 (איור 1 ). את מולקולות צבע להיות ספיגת הבאה ניאון חזק אל תוך התאים (איור 1). טיפולשל תאים עם ligands GPCR מגרה ערוצי GIRK לפתוח. כתוצאה K + בזרימת מחוץ לתא גורמת פוטנציאל הממברנה להיות שלילית יותר את האות ניאון כדי להקטין (איור 1). לכן, תרופות לווסת K + בזרימת דרך ערוץ GIRK ניתן ושם ישקלו לו ויעריכו באמצעות הקורא הצלחת ניאון. שלא כמו אחרים ערוץ יון מבחני המיון, כגון ספיגת ספקטרומטריית אטומית 4 או 5 radiotracer ניתוח, assay ערוץ GIRK ניאון מספק בהליך מיון מהיר, בזמן אמת ולא יקר.

Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

בזמן אמת ההקרנה הליך לזיהוי סמים כי אינטראקציה עם חלבון G-מגודרת פנימה מיישר K<sup&gt; +</sup&gt; (GIRK) ערוצים מתואר. Assay מנצל פוטנציאל רגישות קרום צבעי ניאון כדי למדוד פעילות GIRK הערוץ. טכניקה זו ניתנת להתאמה לשימוש על מספר שורות תאים.

Assay הקרנת פלורסנט לזיהוי מאפננים של ערוצים GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

a-fluorescent-screening-assay-for-identifying-modulators-girk

Research

JoVE Journal

Biology

14.3K Views.  University of South Carolina, School of Medicine. בזמן אמת ההקרנה הליך לזיהוי סמים כי אינטראקציה עם חלבון G-מגודרת פנימה מיישר K + (GIRK) ערוצים מתואר. Assay מנצל פוטנציאל רגישות קרום צבעי ניאון כדי למדוד פעילות GIRK הערוץ. טכניקה זו ניתנת להתאמה לשימוש על מספר שורות תאים.

Video: A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesis וניתוח פונקציונלי של תעלות יונים הביעו Heterologously בתאי יונקים

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

תפוקה גבוהה הקרנת ו biosensing עם פלורסנט ג elegans מתחים

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

המרת ELISA ללכוד Assay Luminex Xmap באמצעות נוגדן Multiplex שיטת הקרנה

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

הקרנת תפוקה גבוהה למאפנני מולקולה הקטנה של ערוצי אשלגן מיישרים פנימה

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

הקרנה פונקציונלית תפוקה גבוהה באמצעות assay בלוציפראז Dual-זוהר תוצרת בית

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

הקרנה ירוקה פלורסנט מבוסס חלבון ביטוי של החלבונים ממברנה ב<em&gt; Coli Escherichia</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Proteoliposome מבוסס הזרימה Assay כדי לקבוע מאפייני מולקולה בודדת של Cl<sup-&gt;</sup&gt; ערוצים ומסועים

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Barcoding הגנטי עם חלבונים ניאון עבור יישומי Multiplexed

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

הערכה של תפקוד כליה דג הזברה באמצעות assay עמילות פלורסנט

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

הקרנת תפוקה גבוהה עבור כימית מאפננים של גני Post-תעתיק מוסדר

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...