Die Forschung hier vorgestellte wurde von Charles River unterstützt.

<ol>
	<li>Jacobson, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+validation+of+diagnostic+assays+for+infectious+diseases.">Principles of validation of diagnostic assays for infectious diseases.</a> Manual of Standards for Diagnostic Tests and Vaccines. , 8-15 (1996).</li><li>Wright, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+standards+for+test+methods+and+reference+sera+for+diagnostic+tests+for+antibody+detection.">International standards for test methods and reference sera for diagnostic tests for antibody detection.</a> Rev. sci. tech. Off. int. Epiz. 17 (2), 527-533 (1998).</li><li>Pepe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Statistical Evaluation of Medical Tests for Classification and Prediction. , (2003).</li><li>Barr, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+interpretation+of+diagnostic+tests+for+feline+immunodeficiency+virus+infection.">Comparison and interpretation of diagnostic tests for feline immunodeficiency virus infection.</a> J. Am. Vet. Med. Assoc. 199, 1377-1381 (1991).</li></ol>

Alle Autoren arbeiten für Charles River RADS, ein führender Anbieter von Tier-Serologie Test-Dienstleistungen und Reagenzien.

Die MFIA Testprozess ist hoch effizient und erfordert weniger Ausrüstung und kleinere Probe und Reagenz Volumen als herkömmliche Singleplex Tests. Die Funktionalität des Multiplex-System gibt dem Benutzer die Flexibilität, um gleichzeitig zu screenen für mehrere Stämme oder Serotypen von gemeinsamen Agenten in Labornagetieren (dh Coronavirus, Parvoviren etc.). Dies ermöglicht es uns, maßgeschneiderte Perlen Panels basieren auf den Bereich von Interesse (zB spezifische Virus-Familie) entwerfen und ist anpassungsfähig an Screening andere Arten von Biomolekülen einschließlich Zytokine und andere Biomarker. Darüber hinaus ermöglicht es uns, mehrere interne Kontrollsystem Assays integrieren, um Probe und System Eignung zu überprüfen und dadurch sicherzustellen, die Genauigkeit der Ergebnisse. Dazu gehören Gewebe-Steuerung und IgG-Anti-Testserum Arten Immunglobulin (αIg) beschichtete Kugel-Sets zu Probe Eignung zu bewerten. Eine Gewebemarkiervorrichtung Steuerwulst erkennt unspezifische Bindung von Serum-Immunglobulin und das αIg Wulst Steuerung bestätigtdass Serum wurde hinzugefügt und enthält eine ausreichende Immunglobulin-Konzentration. Eine weitere Kontrolle Wulst mit Serum Immunglobulin Spezies beschichtet wird, zeigt, dass die markierten Reagentien und Assay Reader ordnungsgemäß funktionieren. Andere im Handel erhältliche Multiplex-Format serologischen Assays (zB Microarrays, ImmunoComb) nicht bieten diese gleiche Ergebnis Bestätigung. Die Fähigkeit zu verengen die mögliche Quelle der Assay Versagen vor Nachprüfung helfen kann ein Ermittler auf Zeit und Material zu sparen. Kritische Aspekte des MFIA sollte vor der Wiederholung einer fehlgeschlagenen Probe bestätigt werden. Da der Test bei Raumtemperatur durchgeführt wird, sollte verifiziert, dass die Temperatur etwa 27 Labor ist ° C ± 2 ° C, höhere Temperaturen führen kann als erwartet Scores für Kontrollen und Proben zu senken. Waschen ist vielleicht der wichtigste Schritt im Assay. Die Versicherung, dass die Testplatte richtig gewaschen, abgetupft und resuspendiert beseitigen könnendie überwiegende Mehrzahl der Abtastfehler aufgrund einer geringen Anzahl Wulst (aufgrund aggregierten Kügelchen) oder unzureichende Reagenzzugabe (Passwort durch Dochtwirkung aus der Testplatte Filterboden). Wir routinemäßig zählen 25 Perlen pro Assay (Agent) und haben keinen statistischen Unterschied in den Ergebnissen durch Zählen höhere Anzahl von Perlen, die auch zu einer längeren Lesezeit für die Platte führen. Wir haben umfassende Validierungsstudien MFIA auf mehrere Arten der am häufigsten verwendeten Versuchstiere (Maus, Ratte, Hamster, Meerschweinchen und Kaninchen) durchgeführt, um die diagnostische Genauigkeit, Reproduzierbarkeit und Robustheit durch das Testen einer großen Anzahl von bekannten positiven und negativen Serumproben zu demonstrieren und Vergleich ihrer ELISA, IFA und MFIA Ergebnisse. Die Nachweisgrenzen (dh Standard-Immunserum Titration Endpunkte) von MFIA vergleichbar waren, und in einigen Fällen übertroffen, wie bei entsprechenden ELISA. Diagnostische Spezifität, mit SPF Nager sera gemessen als 99%, die gesamte Korrespondenz between ELISA und MFIA durchgeführt am bekannten positiven und bekannten negativen Seren war größer als 95%. Zusammenfassend erwiesen diese Ergebnisse, dass eine gute Multiplex MFIA Stellvertreter für den ELISA ist Singleplex, und ist geeignet für die beabsichtigte Verwendung, dh in der Routine serologischen Überwachung von Versuchstieren Kolonien.

multiplexed fluorometric immunoassay testing methodology and troubleshooting

Um die Qualität von Tiermodellen in der biomedizinischen Forschung verwendet sicherzustellen, haben wir eine Reihe von diagnostischen Tests Strategien und Methoden, um festzustellen, ob Tiere zufällige Infektionserregern (Viren, Mykoplasmen, und anderen anspruchsvollen Mikroorganismen) ausgesetzt wurden entwickelt. Infektionen von immunkompetenten Tieren im Allgemeinen vorübergehend, jedoch Serum-Antikörperantworten gegen Infektionen oft innerhalb von Tagen bis Wochen festgestellt werden und bleiben über die gesamte Lebensdauer des Wirts. Serologie ist die primäre diagnostische Methode, mit der Versuchstiere werden überwacht. Historisch die indirekter Enzym-linked Immunosorbent Assay (ELISA) ist die wichtigste Methode zur serologischen Überwachung Screening. Der ELISA ist als Singleplex durchgeführt, wobei ein mikrobielles Antigen-Antikörper-Reaktion pro Vertiefung gemessen. Im Vergleich der MFIA als gemultiplexte Assay durchgeführt. Da die Mikrokügelchen in 100 verschiedene Farbskalen kommen, können bis zu 100 verschiedene Assays simultan durchgeführt werdeneously in einem Mikrotiterplatten. Diese Innovation reduziert die Menge an Serum, Reagenzien und Verbrauchsmaterialien für Routinetests erforderlich, während die Erhöhung der Menge von Informationen aus einem einzigen Test gut erhalten. Darüber hinaus sind wir in der Lage, mehrere interne Kontrollsystem Perlen integrieren, um Probe und System Eignung zu überprüfen und dadurch sicherzustellen, die Genauigkeit der Ergebnisse. Dazu gehören Gewebe-Steuerung und IgG-Anti-Testserum Arten Immunglobulin (αIg) beschichtete Kugel-Sets zu Probe Eignung zu bewerten. Wie in den ELISA und IFA detektiert der Gewebekontrolle unspezifische Bindung von Serum-Immunglobulin. Die αIg Steuerung (Serum-Steuerung) bestätigt, dass das Serum wurde hinzugefügt und enthält eine ausreichende Immunglobulinkonzentration während die IgG-Kontrolle Wulst (System Suitability Kontrolle), mit Serum Arten Immunglobulin beschichtet, zeigt, dass die markierten Reagenzien und Luminex Leser ordnungsgemäß funktionieren.

Watch this Scientific Journal Video about Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting at JoVE.com

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Mit Luminex Corporation xMAP-Mikrokugel-Technologie haben wir die Multiplex Fluorometric Immunoassay (MFIA) für serologischen Überwachung von verschiedenen Labors Tierarten entwickelt. Die MFIA ist eine Suspension Microarray, wo Antigen, Gewebekontrolle oder Immunglobuline kovalent an farbkodierten Polystyrol-Mikrokügelchen verbunden sind. Die MFIA Prüfverfahren sowie verschiedene Themen zur Fehlerbehebung adressiert ist.

Multiplexed Fluorometric Immunoassay Testing Methodology und Fehlerbehebung

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 ...

multiplexed-fluorometric-immunoassay-testing-methodology

Research

JoVE Journal

Biology

28.2K Aufrufe.  Charles River. Mit Luminex Corporation xMAP-Mikrokugel-Technologie haben wir die Multiplex Fluorometric Immunoassay (MFIA) für serologischen Überwachung von verschiedenen Labors Tierarten entwickelt. Die MFIA ist eine Suspension Microarray, wo Antigen, Gewebekontrolle oder Immunglobuline kovalent an farbkodierten Polystyrol-Mikrokügelchen verbunden sind. Die MFIA Prüfverfahren sowie verschiedene Themen zur Fehlerbehebung adressiert ist.

Serie: Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Induktion und Prüfung von Hypoxie in der Zellkultur

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Forschung

Biologie

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Chemisch-Antikörper blockiert Microarray für Multiplex-High-Throughput-Profiling von spezifischen Protein-Glykosylierung bei Complex Samples

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

Polymerase Chain Reaction: Basic Protocol Plus Fehlersuche und Optimierungsstrategien

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

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

Solid Phase Synthesis of a Functionalized Bis-Peptide Using &quot;Safety Catch&quot; Methodology

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

Festphasensynthese eines funktionalisierten Bis-Peptids unter Verwendung von &quot;Safety-Catch&quot;-Methodik

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Design und die Verwendung Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Eine empfindliche und spezifische Quantifizierung Methode zur Bestimmung der Serum Cardiac Myosin Binding Protein-C durch Elektrochemilumineszenz Immunoassay

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

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.

A Guide to Modern Quantitative Fluoreszenz Western Blot mit Strategien zur Fehlersuche

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 ...

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.

Genetische Barcoding mit Fluoreszierende Proteine ​​für Multiplex-Anwendungen

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

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Die Quantifizierung der Proteinexpression und Co-Lokalisation Mit Multiplexed immunhistochemischen Anfärbung und Multispektraltechnik

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

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.

Verfahren und wichtige Optimierungsstrategien für eine automatisierte Kapillar elektrophoretischen basierende Immunoassay-Methode

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