עבודה זו נתמכה על ידי מענק דיסקברי ההפעלה JDS של מדעי הטבע וההנדסה המועצה למחקר (NSERC) של קנדה, NSERC אלכסנדר גרהם בל קנדה מלגות לתארים מתקדמים (דוקטורט) (א &#39;Senatore) וקרן לב ושבץ, גרנט -In-Aid NA # 6284.

<ol>
	<li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> J. Pharmacological and Toxicological Methods. 51, 187-200 (2005).</li><li>Senatore, A., Spafford, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+big+are+key+features+of+an+invertebrate+T-type+channel+(LCav3)+from+the+central+nervous+system+of+Lymnaea+stagnalis.">Transient and big are key features of an invertebrate T-type channel (LCav3) from the central nervous system of Lymnaea stagnalis.</a> Journal of Biological Chemistry. 285, 7447-7458 (2010).</li><li>Ogden, D., Stanfield, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+single+channel+and+whole-cell+recording.">Patch clamp techniques for single channel and whole-cell recording.</a> Microelectrode techniques: the Plymouth workshop handbook. , (1987).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamping:+an+introductory+guide+to+patch+clamp+electrophysiology.">Patch clamping: an introductory guide to patch clamp electrophysiology.</a> , (2003).</li><li>Peng, S. -. Q., Hajela, R. K., Atchison, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+flow-induced+increase+in+inward+Ba2++current+expressed+in+HEK293+cells+transiently+transfected+with+human+neuronal+L-type+Ca2+channels.">Fluid flow-induced increase in inward Ba2+ current expressed in HEK293 cells transiently transfected with human neuronal L-type Ca2+channels.</a> Brain Research. 1045, 116-123 (2005).</li></ol>

אין ניגודי אינטרסים הכריז.

<html> היכולת להקליט כל תא זרמים באמצעות פרוטוקולים תיאר ופתרונות מציין כי transfection היה מוצלח וכי הרצוי מתח מגודרת תעלות יונים או ערוץ מתחמי יון נמצאים עם כמויות משמעותיות על קרום התא. התאים צריכה להיות transfected חיובי (כלומר פלואורסצנטי ירוק), אך לא זרמים לצריבה, זמן נוסף על 2...

<table border="1" style=";text-align:right;direction:rtl"><tr><td> חומר </td><td> חברה </td><td> מספר קטלוגי </td><td> הערה </td></tr><tr><td> pIRES2-EGFP פלסמיד </td><td> Clontech </td><td> 6029-1 </td><td></td></tr><tr><td> חוג זכוכית coverslips </td><td> פישר סיינטיפיק </td><td> 12-545-80 </td><td> מעגלים מס &#39;1-.13 כדי 0.17mm עבה, גודל: 12mm </td></tr><tr><td> מגבר </td><td> אקסון מכשירים </td><td> Axopatch 200B או 700B Multiclamp </td><td></td></tr><tr><td> Data System רכישה </td><td> אקסון מכשירים </td><td> Digidata 1440A </td><td></td></tr><tr><td> Electrophysiology תוכנה </td><td> אקסון מכשירים </td><td> pClamp10.1 </td><td></td></tr><tr><td> פיפטה המניפולטורים </td><td> סאטר Instrument ושות </td><td> MPC-385-2 </td><td></td></tr><tr><td> מיקרוסקופ הפוך Epifluorescence </td><td> Zeiss קנדה </td><td> Axiovert 40 CFL </td><td></td></tr><tr><td> Headstage </td><td> אקסון מכשירים </td><td> CV-7B </td><td></td></tr><tr><td> אלקטרודה Headstage המחזיק </td><td> אקסון מכשירים </td><td> 1-HL-U </td><td></td></tr><tr><td> Microelectrode בעל הקרקע עבור אלקטרודה </td><td> העולם מכשירי דיוק </td><td> MEH3RF 15 </td><td></td></tr><tr><td> Electrophysiology צינורות נימי </td><td> סאטר Instrument ושות </td><td> BF-150-86-15 </td><td> זכוכית בורוסיליקט עם נימה, OD 1.5mm, 0.86mm OD, 15 ס&quot;מ ארוך </td></tr><tr><td> Flaming / בראון micropipette פולר </td><td> סאטר Instrument ושות </td><td> P-97 </td><td></td></tr><tr><td> Micropipette אש לטש </td><td> Narishige </td><td> מיקרו Forge MF-830 </td><td></td></tr><tr><td> זלוף מיקרו התרופה מערכת </td><td> להפוך מדעי </td><td> SmartSquirt8 Valvelink8.2 </td><td></td></tr><tr><td> זלוף והתרופות מערכת </td><td> להפוך מדעי </td><td> Valvelink8.2 עם טפלון שסתומים </td><td></td></tr></table>

optimized transfection strategy for expression and electrophysiological recording of recombinant voltage-gated ion channels in hek-293t cells

בביטוי במבחנה אלקטרו הקלטה של מתח מגודרת ערוצי רקומביננטי יון ב האדם תאים בתרבית כליה עובריים (HEK-293T) היא אסטרטגיית המחקר נמצא בכל מקום. HEK-293T התאים חייב להיות מצופה על coverslips זכוכית בצפיפות מספיק נמוך כדי שהם לא נמצאים במגע זה עם זה על מנת לאפשר הקלטה אלקטרו ללא תופעות בלבול כתוצאה ממגע עם תאים סמוכים. ערוצי transfected צריך גם לבטא עם יעילות גבוהה על קרום התא עבור כל הקלטה תאים תיקון מהדק של זרמים לגילוי מעל רמות הרעש. תעלות יונים Heterologous לעיתים קרובות דורשים תקופות דגירה ארוך 28 מעלות צלזיוס לאחר transfection על מנת להשיג ביטוי הולם קרום, אבל יש להגדיל את ההפסדים של הידבקות התא coverslip ויציבות הממברנה בטמפרטורה זו. כדי לעקוף בעיה זו, פיתחנו אסטרטגיה מותאם transfect צלחת HEK-293T תאים. שיטה זו דורשת כי התאים להיות transfected בבית confluency גבוה יחסית, מודגרות ב 28 ° C לפרקי הדגירה שלאחר transfection כדי לאפשר ביטוי נאות ערוץ יון חלבון. תאים transfected הם מצופה ומשם coverslips זכוכית מודגרות ב 37 ° C למשך מספר שעות, המאפשרת התקשרות תא נוקשה coverslips ו restabilization הממברנה. תאים ניתן להקליט זמן קצר לאחר ציפוי, או ניתן להעביר 28 מעלות צלזיוס במשך הדגירה נוספת. אנו מוצאים כי הדגירה הראשונית ב 28 ° C, לאחר transfection אבל לפני ציפוי, הוא המפתח לביטוי יעיל של תעלות יונים Heterologous שבדרך כלל אינם מבטאים היטב את הממברנה הפלסמטית. Transfected חיובי, תאים בתרבית מזוהים שיתוף הביע eGFP או eGFP הביע מן וקטור bicistronic (למשל pIRES2-EGFP) המכיל את ערוץ יון רקומביננטי cDNA רק נגד הזרם של אתר כניסה פנימי הריבוזום ואת רצף eGFP קידוד. Whole-cell מהדק הקלטה התיקון דורש ציוד מיוחד, בתוספת crafting של אלקטרודות הקלטה מלוטשת בצורת אלקטרודות לקרקע ממשטח הזכוכית בורוסיליקט. משלוח סמים כדי ללמוד את פרמקולוגיה של תעלות יונים יכולה להיות מושגת על ידי ישירות micropipetting סמים לתוך צלחת ההקלטה, או באמצעות microperfusion או זרימת הכבידה מערכות המייצרות זרמים רצוף של פתרון התרופה על תאים מוקלט.

Watch this Scientific Journal Video about Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells at JoVE.com

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

שיטה אמינה היעילה ביותר<em&gt; במבחנה</em&gt; הביטוי הקלטה אלקטרו הבאות של רקומביננטי מתח מגודרת תעלות יונים של האדם תאים בתרבית כליה עובריים (HEK-293T).

Transfection אסטרטגיה אופטימיזציה עבור הביטוי הקלטה של ​​אלקטרו Voltage-מגודרת ערוצי רקומביננטי יון ב HEK-293T תאים

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

optimized-transfection-strategy-for-expression-electrophysiological

Research

JoVE Journal

Biology

32.2K Views.  University of Waterloo. שיטה אמינה היעילה ביותר במבחנה הביטוי הקלטה אלקטרו הבאות של רקומביננטי מתח מגודרת תעלות יונים של האדם תאים בתרבית כליה עובריים (HEK-293T).

Video: Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

הצמד תיקון הקלטה של ​​תעלות יונים מתבטא ביציות Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

מסיק והכנת תסיסנית עוברים הקלטה אלקטרו ונהלים אחרים

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

הקלטה של ​​אלקטרו תסיסנית עובריים

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

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

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

Mutagenesis וניתוח פונקציונלי של תעלות יונים הביעו Heterologously בתאי יונקים

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

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

הצמד תיקון וטכניקות זלוף עבור לימוד תעלות יונים מתבטא Xenopus ביציות

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

כינונה מחדש של חלבון טראנסממברנלי, יון ערוץ מתח מגודרת, KvAP, לענק Unilamellar שלפוחית ​​למיקרוסקופי וצמד תיקון ללימודים

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

רב תכליתיים, Micropipette מבוסס שיטה להתאגדות וגירוי של ערוצים בקטריאלי Mechanosensitive יון בbilayers ממשק אגל

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

ביטוי תשואה גבוהה של חלבונים אנושיים רקומביננטי עם transfection חולף של תאי HEK293 בהשעיה

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

קביעת פני התא יחסית והביטוי סה&quot;כ של תעלות יונים רקומביננטי שימוש cytometry זרימה

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

ביטוי חולף ואת סלולר לוקליזציה של חלבונים רקומביננטי תאים בתרבית חרקים

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Transfection אסטרטגיה אופטימיזציה עבור הביטוי הקלטה של ​​אלקטרו Voltage-מגודרת ערוצי רקומביננטי יון ב HEK-293T תאים

Transfection אסטרטגיה אופטימיזציה עבור הביטוי הקלטה של אלקטרו Voltage-מגודרת ערוצי רקומביננטי יון ב HEK-293T תאים