1. Antikörper-Capture auf High Capacity Magnetic Protein A oder Protein G Magnetic Beads <ol><li> Einheitlich resuspendieren magnetische Kügelchen durch leichtes Schütteln. Halten Sie die Suspension gleichmäßig, wenn Aliquotierung Perlen. </li><li> Je 50 ul Kügelchenaufschlämmung in ein 1,5 ml Mikrozentrifugenröhrchen. Legen Sie in dem magnetischen Ständer für 10 Sekunden. Sie vorsichtig den Speicherpuffer zu entfernen. </li><li> In 250 ul Antikörper-Bindungspuffer. </li><li> Mischen Sie und legen Sie in dem magnetischen Ständer für 10 Sekunden. Sie vorsichtig den Bindungspuffer zu entfernen. </li><li> 1,0 ml der Probe, die 50-100 ug Antikörper an die Kügelchen. Die Proben können Antikörper oder Antikörper in Zellmedien gereinigt werden. </li><li> Mix Probe für 60 min bei RT mit einem Vortex-Mischer oder End-Over-End-Mixer. </li><li> Das Röhrchen in den magnetischen Ständer für 10 Sekunden und entfernen Sie den Überstand. </li><li> In 250 ul Antikörper-Bindungs ​​/ Waschpuffer und mischen. Legen Sie in dem magnetischen Ständer für 10 Sekunden und entfernen Bindung / Waschpuffer. Wiederholen Sie diesen Vorgang step für insgesamt zwei Waschungen. </li><li> Fahren Sie mit Abschnitt 2 Etikett unter Verwendung von Antikörpern Aminchemie oder in Abschnitt 3 zur Kennzeichnung von Antikörpern Thiolchemie. </li></ol> 2. Antikörpermarkierung Mit Amine Chemie <ol><li> Konjugatantikörper <ol><li> 100 l Amin Konjugationspuffer an die Kügelchen. </li><li> Lösen die aminreaktive Fluoreszenzfarbstoffe (AlexaFluor 532-SE) bei 10 mg / ml durch Zugabe von 100 &amp; mgr; l Dimethylsulfoxid (DMSO) bis 1,0 mg Farbstoff. Mix von Verwirbelung. Machen Sie diese Lösung kurz vor dem Gebrauch. </li><li> Hinzufügen, 2,5 ul aminreaktiven Farbstoff für 100 ug Antikörper. Anmerkung: In der Regel eine 5-20 molarer Überschuß an Reaktivfarbstoff empfohlen. Jedoch zugegebene Menge an Reaktivfarbstoff auf die Reaktions empirisch optimiert werden muss, je nach gewünschten Farbstoff-Antikörper-Verhältnis und intrinsische Antikörpereigenschaften. </li><li> Mix Probe für 60 min bei RT mit einem Vortex-Mischer oder End-Over-End-Mixer. Stellen Sie sicher, dass die Perlen in suspensio bleibenn. </li><li> Das Röhrchen in den magnetischen Ständer für 10 Sekunden und entfernen Sie den Überstand. </li><li> In 250 ul Antikörper-Bindungs ​​/ Waschpuffer und mischen. Legen Sie in dem magnetischen Ständer für 10 Sekunden. Entfernen und entsorgen Bindung / Waschpuffer. Wiederholen Sie diesen Schritt für insgesamt zwei Wäschen. </li></ol></li><li> Antikörper Rettung <ol><li> In 50 ul Elutionspuffer an die Kügelchen. </li><li> Mischen Sie für 5 min bei RT mit einem Vortex-Mischer oder End-Over-End-Mixer. Stellen Sie sicher, dass die Perlen in Suspension bleiben. </li><li> Das Röhrchen in den magnetischen Ständer für 10 Sekunden. Entfernen eluierten Probe und Transfer in ein neues Mikrozentrifugenröhrchen, die 5 &amp; mgr; l Neutralisationspuffer. </li><li> Wiederholen Sie den Vorgang noch einmal und bündeln die eluierten Proben. </li><li> Quantifizieren der Antikörperkonzentration und Farbstoff-zu-Antikörper-Verhältnis, wie in Abschnitt 4 beschrieben. </li></ol></li></ol> 3. Antikörpermarkierung Mit Thiolchemie <ol><li> Antikörper Reduction <ol><li> In 250 ulvon Thiolkonjugation Puffer und Mix. Das Röhrchen wird in dem magnetischen Ständer für 10 Sekunden. Entfernen Sie den Puffer zu verwerfen. Zweimal wiederholen Sie diesen Schritt. </li><li> 100 l Thiolkonjugation Puffer. </li><li> Hinzufügen, Dithiothreitol (DTT) auf eine Endkonzentration von 2,5 mM. </li><li> Mix Probe für 60 min bei RT mit einem Vortex-Mischer oder End-Over-End-Mixer. Stellen Sie sicher, dass die Perlen in Suspension bleiben. </li><li> Das Röhrchen wird in dem magnetischen Ständer für 10 Sekunden und den Puffer zu verwerfen. </li><li> In 250 ul Thiolkonjugation Puffer und mischen. Das Röhrchen wird in dem magnetischen Ständer für 10 Sekunden. Entfernen Sie den Puffer zu verwerfen. Wiederholen Sie diesen Schritt für insgesamt zwei Wäschen. </li><li> 100 l Thiolkonjugation Puffer. </li></ol></li><li> Konjugatantikörper <ol><li> Man löst den Thiol-reaktiven Farbstoff bei 10 mg / ml durch Zugabe von 100 &amp; mgr; l DMSO zu 1,0 mg Farbstoff. Mix von Verwirbelung. Machen Sie diese Lösung kurz vor dem Gebrauch. </li><li> Hinzufügen, 2,5 ul thiol-Reaktivfarbstoff für 100 ug antiKörper. Anmerkung: In der Regel eine 5-20 molarer Überschuß an Reaktivfarbstoff empfohlen. Jedoch zugegebene Menge an Reaktivfarbstoff auf die Reaktions empirisch optimiert werden muss, je nach gewünschten Farbstoff-Antikörper-Verhältnis und intrinsische Antikörpereigenschaften. </li><li> Mix Probe für 60 min bei RT mit einem Vortex-Mischer oder End-Over-End-Mixer. Stellen Sie sicher, dass die Perlen in Suspension bleiben. </li><li> Das Röhrchen wird in dem magnetischen Ständer für 10 Sekunden und entfernen Sie den Überstand. </li><li> In 250 ul Thiolkonjugation Puffer und mischen. Legen Sie in dem magnetischen Ständer für 10 Sekunden und den Puffer zu entfernen. Wiederholen Sie diesen Schritt für insgesamt zwei Wäschen. </li></ol></li><li> Eluieren Antikörper <ol><li> In 50 ul Elutionspuffer an die Kügelchen. </li><li> Mischen Sie für 5 min bei RT mit einem Vortex-Mischer oder End-Over-End-Mixer. Stellen Sie sicher, dass die Perlen in Suspension bleiben. </li><li> Das Röhrchen in den magnetischen Ständer für 10 Sekunden. Entfernen Sie eluierten Probe und in ein neues Mikrozentrifugenröhrchen, das 5 ul neutralization Puffer. </li><li> Wiederholen Sie den Vorgang noch einmal und bündeln die eluierten Proben. </li><li> Quantifizieren der Antikörperkonzentration und Farbstoff-zu-Antikörper-Verhältnis, wie in Abschnitt 4 beschrieben. </li></ol></li></ol> 4. Berechnen Dye-zu-Antikörper-Verhältnis <ol><li> Messen der Extinktion des Antikörper-Farbstoff-Konjugat bei 280 nm (A280) und an der &amp; lambda; max für den Farbstoff (Amax). </li><li> Berechnen Sie die Antikörperkonzentration: Antikörper - Konzentration (mg / ml) = A280 - (A max × CF) / 1,4 wo CF = Korrekturfaktor des Farbstoffes (vom Hersteller zur Verfügung gestellt). </li><li> Berechnen Sie den Farbstoff-zu-Antikörper-Verhältnis: Farbstoff-zu-Antikörper - Verhältnis (DAR) = (a max × 150.000) / Ab - Konzentration (mg / ml) × ε Farbstoff wobei ε = Farbstoff der Extinktionskoeffizient des Farbstoffs an seinem Absorptionsmaximum und das Molekulargewicht des Antikörpers = 150000 Da. </li></ol>

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Authors are employees of Promega Corporation.

Das Ziel dieser Studie war es, ein Verfahren zu entwickeln, Antikörper, die in dem Zellmedium in geringen Konzentrationen, mit einer Vielzahl von kleinen Molekülen zu markieren. Ein derartiges Verfahren, eine große Anzahl von Antikörpern, während der frühen Phasen der Antikörper Entdeckung ermöglicht, mit kleinen Molekülen und gesiebt unter Verwendung eines biologisch relevanten Test gekennzeichnet werden. Ein solcher Assay ist der Rezeptor-vermittelten Internalisierung Antikörper Assay wo Internalisierung zwi...

Antikörper , markiert mit kleinen Molekülen sind vielleicht die am häufigsten verwendeten Reagenzien in der Biologie 1,2. Antikörper mit Fluoreszenzfarbstoffen markierte und Biotin werden in großem Umfang in der Bildgebung, Immunoassays, Durchflusszytometrie, Western - Blots und Immunpräzipitation unter anderen Anwendungen 3-6. Radioaktiv markierte Antikörper 3,7 finden umfangreiche Verwendung in der Bildgebung und Therapie, markierte Antikörper mit Zytostatika (ADCs) bieten neue Möglichkeiten für die Behandlung von Krebs und zwei ADCs wurden bereits für den therapeutischen Einsatz 8 genehmigt. Trotz einer umfassenden Nutzung haben sich die Verfahren zur Markierung von Antikörpern blieb überraschenderweise unverändert und beinhalten typischerweise mehrere Reaktionen und Entsalzung Schritte 9-12. Lösungsmethoden arbeiten sehr gut in Fällen, in denen nur wenige Antikörper müssen gekennzeichnet werden und in gereinigter Form zur Verfügung stehen hoch in hoher Konzentration und in ausreichender Menge. Jedoch für neuere Anwendungen wie ADCs gibt es einAntikörper müssen in der frühen Stufe Hybridom zu etikettieren , so dass sie für biologisch relevante Eigenschaften gescreent werden, beispielsweise rezeptorvermittelte Internalisierung Antikörper 13-16. Bei dem Hybridom Stadium sind Probenvolumina begrenzt, Antikörper werden in niedrigen Konzentrationen exprimiert und eine Anzahl von Proben sind groß, daher Lösung auf Basis Markierungsmethoden sind nicht geeignet. Zur Vereinfachung und den Durchsatz der traditionellen Antikörpermarkierung Methoden zu verbessern, einige alternative Ansätze wurden 17,18 vorgeschlagen. Ein Ansatz ist es nicht magnetischen Protein-A-Affinitätskügelchen in kleine Säulen gepackt zu verwenden, um Antikörper, gefolgt von der Markierungsreaktion und Elution von markiertem und gereinigtem Protein erfassen. Dieses Verfahren verwendet werden, die Antikörper direkt aus Zellmedien zu beschriften, jedoch kann die Verwendung von Säulen umständlich sein. Ein magnetisches bead basierte Methode wurde kürzlich berichtet worden , 19 daß die Verwendung von Spalten eliminiert und verbessert den Durchsatz , aber wegender begrenzten Antikörperkapazität der Beads Bindung nur Nanogramm bis niedrigen Mikrogramm-Mengen der Antikörper markiert werden. Wir haben vor kurzem entwickelt und verwendet hoher Kapazität magnetischen Protein A und Protein G - Kügelchen (&gt; 20 mg Human - IgG / ml des abgesetzten Kügelchen) 20 - Antikörpern in Zellmedien mit kleinen Molekülen zu markieren. Die hohe Kapazität der Kügelchen ermöglicht zehn bis hundert Mikrogramm Antikörper bequem gekennzeichnet werden und die schnelle magnetische Reaktion der Kügelchen vereinfacht die Handhabung und Verarbeitung einer großen Anzahl von Proben in parallel. Verwendung von Fluoreszenzfarbstoffen als Surrogate für kleine Moleküle zeigen wir, dass das Verfahren mit dem Amin und Thiol Markierungschemie kompatibel und bietet eine hohe Gewinnungsraten von markierten und sehr reine Antikörper. Dieses Protokoll und die zugehörigen Video beschreiben On-Bead-Kennzeichnung von Maus-Antikörper, die in den Zellmedien Magnetic Protein A und Protein G-Kügelchen verwendet wird. Das Protokoll istgliedert sich in vier Abschnitte: Abschnitt 1 beschreibt die Erfassung von Antikörpern auf den Wulst aus biologischen Proben. Nach Erfassung der Markierung von Antikörpern mit Fluoreszenzfarbstoff Aminchemie oder Thiolchemie verwendet, ist in den Abschnitten 2 und Abschnitt 3 beschrieben sind. Schließlich Abschnitt 4 beschreibt das Verfahren zur Berechnung der Antikörperkonzentration und der Farbstoff-Antikörper-Verhältnis.

<table border="0" cellpadding="0" cellspacing="0" width="592">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein A Beads</td>
			<td style="width:103px;">Promega Corporation</td>
			<td style="width:119px;">&#160;G8781</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein G Beads</td>
			<td style="width:103px;">Promega Corporation&#160;</td>
			<td style="width:119px;">&#160;G7471</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-SE (Succinimidyl Ester)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-ME (Malemide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A10255</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 647-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20347</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescein-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">F-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic Stand</td>
			<td style="width:103px;">Promega</td>
			<td style="width:119px;">Z5332</td>
			<td></td>
		</tr>
	</tbody>
</table>

antibody labeling with fluorescent dyes using magnetic protein a and protein g beads

Antikörper, markiert mit kleinen Molekülen, wie fluoreszierende Farbstoffe, Zytostatika, und radioaktive Tracer sind wesentliche Instrumente in der biomedizinischen Forschung, der Immundiagnostik und in jüngerer Zeit als therapeutische Wirkstoffe. Herkömmliche Verfahren zur Markierung von Antikörpern mit kleinen Molekülen erfordern Antikörper in relativ hoher Konzentration, beinhalten mehrere Dialyseschritte gereinigt und haben eine begrenzte Durchsatz. Jedoch mehrere Anwendungen, einschließlich dem Gebiet der Antikörper-Wirkstoff-Konjugate (ADCs), werden von den neuen Methoden profitieren, die Markierung von Antikörpern direkt von Zellmedien ermöglicht. in biologisch relevanten Assays gescreent werden, beispielsweise der Rezeptor-vermittelten Internalisierung Antikörper-Assay im Falle von ADCs Solche Verfahren können Antikörper ermöglichen. Hier beschreiben wir ein Verfahren (on-bead-Methode), die Etikettierung von kleinen Mengen von Antikörpern direkt aus Zellmedien ermöglicht. Dieser Ansatz nutzt hohe Kapazität magnetischen Protein A und Protein G Affinitätsbeads Antikörper zu erfassenaus den Zellmedien durch Markierung mit kleinen Molekülen, gefolgt entweder Amin- oder Thiol-Chemie und anschließende Elution der markierten Antikörper verwendet. Unter Fluoreszenzfarbstoffe als Surrogate für kleine Moleküle, zeigen wir die On-Bead-Kennzeichnung von drei verschiedenen Maus-Antikörper direkt aus Zellmedien sowohl Amin und Thiol Markierungschemie verwendet wird. Die hohe Bindungsaffinität von Antikörpern an Protein A und Protein G sorgt für eine hohe Wiederfindungsraten sowie hoher Reinheit der markierten Antikörper. Darüber hinaus ermöglicht die Verwendung von magnetischen Kügelchen mehrere Proben manuell, wodurch deutlich Kennzeichnungs Durchsatz verbessert behandelt werden.

Eine schematische für Antikörper mit kleinen Molekülen unter Verwendung von hoher Kapazität magnetischen Protein A und Protein G - Kügelchen Kennzeichnung ist in Abbildung 1 dargestellt. Antibodies auf dem magnetischen Protein G - Kügelchen eingefangen kann mit kleinen Molekülen markiert werden, beispielsweise Fluoreszenzfarbstoffe, entweder Aminchemie , die Etiketten primäre Amine der Lysin Aminosäuren oder unter Verwendung von Thiol-Chemie, die bei den reduzie...

Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Die on-bead-Verfahren zum Nachweis von Antikörpern mit kleinen Molekülen Kennzeichnung ermöglicht die Kennzeichnung von einer geringen Menge an Antikörper direkt aus Zellmedien. Dieses Verfahren ist kompatibel mit Amin und Thiolchemie und kann mehrere Proben parallel, manuell oder mit automatisierten Systemen verarbeiten.

Antikörpermarkierung mit Fluoreszenzfarbstoffen unter Verwendung von magnetischen Protein A und Protein G-Kügelchen

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

antibody-labeling-with-fluorescent-dyes-using-magnetic-protein

Research

JoVE Journal

Immunology and Infection

11.7K Aufrufe.  Promega Corporation.  Die on-bead-Verfahren zum Nachweis von Antikörpern mit kleinen Molekülen Kennzeichnung ermöglicht die Kennzeichnung von einer geringen Menge an Antikörper direkt aus Zellmedien. Dieses Verfahren ist kompatibel mit Amin und Thiolchemie und kann mehrere Proben parallel, manuell oder mit automatisierten Systemen verarbeiten. 

Serie: Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Spezielle Kennzeichnung von HIV-1 positiven Zellen mit Hilfe eines Rev-abhängigen Lentivirale Vector mit dem Ausdruck des Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Forschung

Immunologie und Infektion

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Ein Calcium Biolumineszenzassay für die funktionelle Analyse von Mosquito ( Aedes aegypti) Und Tick ( Rhipicephalus microplus) G-Protein gekoppelten Rezeptoren

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

Darmkrebs Zelloberflächenprotein Profiling mit einem Antikörper Microarray und Fluoreszenz Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Proteinfehlfaltungen Cyclic Amplification von Prionen

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Überwachen von Änderungen in Membran-Polarity, Membrane Integrity und intrazelluläre Ionenkonzentrationen in<em&gt; Streptococcus pneumoniae</em&gt; Verwenden von Fluoreszenzfarbstoffen

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-Immunopräzipitation der Maus Mx1 Protein mit dem Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

Impfen<em&gt; Anopheles gambiae</em&gt; Mosquitoes mit Perlen der Melanisierung Immunantwort zu induzieren und Messen

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Sezieren Signalisierung Multi-Protein-komplexe durch eine Bimolekulare Ergänzung Affinitätsreinigung (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Ein Protein-Microarray-Assay für die serologische Bestimmung der Antigen-spezifische Antikörper Antworten folgende Clostridium Difficile Infektion

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

Antikörpermarkierung mit Fluoreszenzfarbstoffen unter Verwendung von magnetischen Protein A und Protein G-Kügelchen

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