עבודה זו מומנה על ידי Luminex Corporation.

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עבודה זו נעשתה על Luminex חברת הציוד מיוצרים ב Luminex Corporation. R &amp; D מערכות &amp; EMD Millipore הם שותפים אסטרטגיים של Luminex Corporation; רישיון לפתח ולמסחר Xmap זמנית מבחני מבוסס.

המרה של assay ELISA לפלטפורמה Luminex Xmap יכול להיות פשוט כמו החלפת streptavidin peroxidase חזרת (SA-HRP) ב טיפוסית ערכת ELISA עם streptavidin phycoerythrin (SA-PE), ואופטימיזציה של הביצועים. למי שרוצה ליצור immunoassay Xmap מהיסוד, זה יכול להיות מושלם עם פרוטוקול ברור כי גם מאפשר הערכה מהירה, זמנית של זוגות נוגדנים. ריאגנטים לשנת assay Xmap היו מוכנים בקלות באמצעות נוגדן Xmap ערכת צימוד לזוג נוגדנים ללכוד המיועדים MagPlex microspheres ריכוז נמוך. השימוש microspheres ריכוז נמוך מפחית את העלות של פיתוח assay תוך מתן ביצועים assay אותו microspheres ריכוז גבוהה יותר. משך הזמן הנדרש כדי להכין יחד MagPlex microspheres הוא כ 3 שעות, שזה הרבה יותר מהר מאשר 22 - 24 שעות נדרש המעיל גם צלחת ELISA, ואחריו טיפולשל בארות מצופים. הביצועים של assay Xmap הוא גם עדיפה על ELISA מבחינת מגבלה של זיהוי (&lt;4 pg / mL לעומת&gt; 31 pg / mL) ודינמי טווח (&lt;4 pg / mL ל&gt; 8000 pg / mL לעומת 16 pg / mL עד 1000 pg / ml). קוראים פלייט יש מגוון מצומצם OD אשר הוא או 3 או 4 OD, להגביל את הגבול העליון של טווח דינמי assay. אין ספק, לא כל הנוגדנים יעבדו במתכונת ELISA ולא כל הנוגדנים שפועלים היטב ב ELISA ניתנות להעברה בקלות לתבנית assay Xmap. עם זאת, מאז מבחני Xmap ניתן מרובב (כלומר, להפעיל בו זמנית), ניתן להעריך ללכוד כמה שילובים ואת הנוגדנים לזיהוי במקביל לזהות את הצמד הטוב ביותר לשימוש עבור assay. תהליך זה חוסך זמן משמעותי ריאגנטים לעומת הליך פיתוח ELISA, אשר מוגבל להערכת זוג אחד בכל פעם. שניים או יותר זוגות הנוגדנים צריך לבצע שקול, פרמטרים אחרים של assay יכוללשקול לקבוע התאמתו של זוג (למשל, זמינות, עלות וכו &#39;). בנוסף לביצועים assay גמישות משופרת עם assay Xmap, יש גם חיסכון משמעותי בעלויות. כמות מומלצת של נוגדנים נדרש מעיל גם יחידה של צלחת ELISA היא 400ng, בעוד הכמות הדרושה של חרוזים המשמשים 1 היטב assay Xmap הוא כ 7.5 ננוגרם. לפיכך, כמות הנוגדנים הדרוש 1 ELISA גם תספק יותר מ -50 תוצאות הבדיקה, אם נעשה שימוש assay Xmap. עבור יישומים הכוללים דגימות יקרות, Xmap יש גם יתרון משמעותי. היקף המדגם מומלץ ELISA הוא 100 μL ואילו נפח הנדרש assay Xmap יכול להיות חצי מזה, או פחות. לסיכום, המרה של assay ELISA לפלטפורמה Luminex Xmap הוא לא מסובך, יעיל ועלות חיסכון, תוך הפקת assay עם טווח דינמי רגישות מעולה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> תאור </td><td> מוכר </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> האדם TNF-α/TNFSF1A DuoSet ערכת ELISA </td><td> מחקר ופיתוח מערכות </td><td> DY210 </td><td> כולל חד שבטיים ו ביוטין נוגדנים polyclonal יחד ו רקומביננטי סטנדרטיים TNF-α חלבון </td></tr><tr><td> נוגדן חד שבטי כדי TNF-α </td><td> Abcam </td><td> Ab18696 </td><td> לכידת נוגדנים, שיבוט CH8820 </td></tr><tr><td> נוגדן חד שבטי כדי TNF-α </td><td> Abcam </td><td> Ab16166 </td><td> ביוטין מצמידים נוגדן איתור, שיבוט AS1 </td></tr><tr><td> חלבון TNF אלפא </td><td> Abcam </td><td> Ab9642 </td><td> רקומביננטי TNF-α חלבון רגילה </td></tr><tr><td> נוגדן חד שבטי כדי TNF-α </td><td> לא זמיןovus </td><td> NBP1-50115 </td><td> לכידת נוגדנים, שיבוט 4H31 </td></tr><tr><td> נוגדן חד שבטי כדי TNF-α </td><td> נובוס </td><td> NB100-78162 </td><td> ביוטין מצמידים נוגדן איתור, שיבוט MAb11 </td></tr><tr><td> חלבון TNF אלפא </td><td> נובוס </td><td> NBC1-18460 </td><td> רקומביננטי TNF-α חלבון רגילה </td></tr><tr><td> נוגדן חד שבטי כדי TNF-α </td><td> EMD Millipore </td><td> MAB1141 </td><td> לכידת נוגדנים, לשבט 3C7.2 </td></tr><tr><td> נוגדן Polyclonal כדי TNF-α </td><td> EMD Millipore </td><td> 654250 </td><td> איתור polyclonal הארנב נוגדן </td></tr><tr><td> Streptavidin-Phycoerythrin </td><td> אזוב </td><td> SAPE-001 </td><td> כתב מגיב פלורסנט עבור assay Xmap Luminex </td></tr><tr><td> MAGPIX w / Software xPONENT </td><td> Luminex Corporation </td><td> תואר שניGPIX-XPONENT </td><td> Luminex Instrument </td></tr><tr><td> Xmap נוגדן צימוד (ABC) ערכת </td><td> Luminex Corporation </td><td> 40-50016 </td><td> כולל מגיב EDC, Sulfo-NHS מגיב, מאגר ההפעלה, מאגר הכביסה, 1.5 התגובה צינורות מ&quot;ל, ו בטפי להחדרת נוזלים חד פעמיים </td></tr><tr><td> MagPlex microspheres, ריכוז נמוך </td><td> Luminex Corporation </td><td> MC10012-ID, MC10013-ID, MC10014-ID, MC10015-ID </td><td> ריכוז נמוך חרוזים (@ 2.5 x 10 6 חרוז / mL) </td></tr><tr><td> EZ-Link Sulfo-NHS-LC-ביוטין </td><td> תרמו פישר </td><td> PI-21335 </td><td> Biotinylation ערכת זיהוי הנוגדן ללא שינוי </td></tr><tr><td> טקאן אינסופי F200 Reader </td><td> טקאן </td><td></td><td> ELISA צלחת הקורא </td></tr><tr><td> פוספט שנאגרו מלוחים </td><td> Sigma-Aldrich </td><td> P-3688 </td><td> 1% PBS-BSA, Assay Buffer </td></tr><tr><td> אחד פיינט נקיים קולי קומפקטי, 115 VAC </td><td> קולמן בן הזוג </td><td> WU-08849-00 </td><td> לייצר תדר הפעלה יעילה של kHz 55 </td></tr><tr><td> Tube מפריד מגנטי </td><td> Luminex Corporation </td><td> CN-0288-01 </td><td> להפרדה חד צינור 1.5mL המגנטי צעדים לשטוף צימוד </td></tr><tr><td> לוח מגנטי מפריד </td><td> Luminex Corporation </td><td> CN-0269-01 </td><td> להפרדה 96 היטב צלחת מגנטית בצעדים לשטוף assay </td></tr></tbody></table>

conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method

Assay האנזים צמוד immunosorbent (ELISA) כבר זמן רב את הכלי העיקרי לגילוי של analytes של עניין דגימות ביולוגיות הן למחקר מדעי החיים ודיאגנוסטיקה קלינית. עם זאת, ELISA יש מגבלות. הדבר מתבצע בדרך כלל microplate 96-היטב, בארות מכוסים נוגדנים ללכוד, הדורש כמות גדולה יחסית של המדגם לתפוס אנטיגן של עניין. שטח פנים גדול של בארות מחייב הידרופובי של נוגדנים ללכוד יכול להוביל גם הרקע הלא ספציפית מחייב יותר. בנוסף, רוב ELISAs להסתמך על הגברה האנזים בתיווך של אות על מנת להשיג רגישות סבירה. הגברה כזו היא לא תמיד ליניארית תוצאות ולכן ניתן ליצור הטיה. ב -15 השנים האחרונות, התפתחה טכנולוגיה חדשה המציעה את היתרונות של ELISA, אך גם מאפשר קצב העברת נתונים גבוהה יותר, גמישות משופרת, נפח דגימה מופחת, ובעלות נמוכה יותר, עם וו דומהrkflow 1, 2. Luminex Xmap הטכנולוגיה היא (חרוז) microsphere מערך פלטפורמה המאפשרת הן monoplex ואת מבחני זמנית כי ניתן להחיל את החלבון הן חומצות גרעין ויישומים 3-5. החרוזים יש נוגדנים ללכוד משותקת קוולנטית על שטח פנים קטן יותר, המחייב נוגדנים ללכוד פחות כרכים מדגם קטן יותר, בהשוואה ל ELISA, ולא ספציפית מחייב מצטמצם משמעותית. כרכים מדגם קטנים יותר חשובים בעבודה עם דגימות מגבילות כמו הנוזל השדרתי, נוזל סינוביאלי, וכו &#39;6. ריבוב assay תצמצם עוד יותר את נפח דרישות לדוגמה, המאפשר מספר רב של תוצאות ממדגם אחד. שיפורים שנעשו לאחרונה על ידי Luminex כוללים: המערכת החדשה MAGPIX, קטן יותר, זול יותר, קל יותר לשימוש Analyzer, ריכוז נמוך מגנטי MagPlex microspheres אשר מבטל את הצורך צלחות הסינון יקרים לבוא בריכוז עובד מתאים יותר לפיתוח assay ונמוך התפוקה יישומים וכן את הצימוד נוגדן Xmap (ABC) קיט, הכולל פרוטוקול, חומרים כימיים, וחומרים מתכלים הדרושים צימוד חרוזים כדי נוגדנים לכידתו של עניין. (ראה סעיף חומרים לרשימה מפורטת של תכולת הערכה). בניסוי זה, אנו ממירים את assay מראש אופטימיזציה ELISA עבור ציטוקינים TNF-alpha לפלטפורמה Xmap ולהשוות את הביצועים של שתי השיטות 7-11. TNF-alpha הוא סמן ביולוגי המשמש למדידת תגובות דלקתיות בחולים עם מחלות אוטואימוניות. נתחיל צימוד ללכוד ארבעה נוגדנים מועמד ארבע קבוצות microsphere או אזורים שונים. כאשר מערבבים יחד, אלה ארבע קבוצות לאפשר בדיקות סימולטני של כל ארבעת המועמדים עם ארבעה נוגדנים זיהוי נפרדים כדי לקבוע זוג נוגדנים הטובה ביותר, ריאגנטים חיסכון, מדגם וזמן. שני מבחני Xmap בנויים כך עם שני זוגות נוגדנים האופטימלית ביותר והביצועים שלהםבהשוואה לזו של assay ELISA המקורי לגבי עוצמת האות, טווח דינמי, ורגישות.

Watch this Scientific Journal Video about Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method at JoVE.com

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

ELISA ניתן להמיר בקלות assay Luminex Xmap, ובאמצעות היתרונות של נוגדנים, ריבוב כמה יכול להיות מוקרן בו זמנית לזהות נוגדנים זוג אופטימלי, והתוצאה היא רגישות מוגברת טווח דינמי, תוך הפחתת עלות assay.

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 ...

conversion-capture-elisa-to-luminex-xmap-assay-using-multiplex

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JoVE Journal

Biology

43.4K Views.  Luminex Corporation. ELISA ניתן להמיר בקלות assay Luminex Xmap, ובאמצעות היתרונות של נוגדנים, ריבוב כמה יכול להיות מוקרן בו זמנית לזהות נוגדנים זוג אופטימלי, והתוצאה היא רגישות מוגברת טווח דינמי, תוך הפחתת עלות assay.

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

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

לייזר Microdissection לכידה של רקמות היונקים

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Cell Capture Using a Microfluidic Device

תא לכידת באמצעות מכשיר microfluidic

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

שיטת מכתים נוגדן שלם הר ב צ&#39;יק

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi הקרנת לזהות פנוטיפים Postembryonic ב ג elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

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 Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

שלב נוזלי זיקה לכידת Assay באמצעות חרוזים מגנטיים ללמוד חלבונים אינטראקציה: דוגמה poliovirus-Nanobody

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

שיטה לבחירת Aptamers מיתוג מבנה יישומית לColorimetric זהב Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

זיהוי של זוגות קינאז-מצע שימוש הקרנת תפוקה גבוהה


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

הקרנה רב-אנזים באמצעות מערכת הקרנת אנזים הגנטי תפוקה גבוהה

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...