We wish to thank the Calcium Imaging Core Facility at CMS for their help and guidance in image capture and analysis. This study was supported by the U.S. National Institutes of Health (NIH) HL102208 to N.A.B. This approach was originally pioneered by Holger Lorenz and Jennifer Lippincott-Schwartz at NIH.

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The authors declare no conflict of interest.

ההתמצאות וטופולוגיה הנכונות של חלבונים בממברנה היא חיוני לתפקוד התקין שלהם. למרות החשיבות של הבנת טופולוגיה חלבון קרום, יש חלבונים רבים שעבורם נתונים כאלה חסרים לחלוטין. FPP מספק דרך קלה ויעילה לקביעת טופולוגיה חלבון קרום, ואחד שיכול להתבצע על ידי רוב המעבדות. גישת FPP ...

ממברנות הפלזמה, כמו גם את הקרומים תאיים הרבים, שמשו כחיץ המפריד בין שני תאים מימיים. במקרה של קרום הפלזמה, ההפרדה היא בין החוץ ופנים של התא; לאברונים תוך-תאיים הוא בין הציטופלסמה ולומן אברון. לדוגמא, קרום reticulum endoplasmic (ER) מפריד בין סביבת חמצון בתוך לומן של ER מcytosolic צמצום הסביבה 1. חלבוני קרום מסונתזים על ריבוזומים הקשורים ER, ולהשיג טופולוגיה הסופית שלהם בתוך הקרום ER 2. רכישת אוריינטצית קרום המתאימה וטופולוגיה לחלבונים היא חיונית לתפקוד הנורמלי שלהם. טופולוגיה הנכונה מאפשרת תחומים רלוונטיים של חלבונים בממברנה כדי אינטראקציה עם השותפים המחייבים שלהם, הוא מאפשר לאחר translational שינויים קריטיים להתרחש, ובמקרה של חלבוני קרום פלזמה, מאפשר לתא לקיים אינטראקציה עם ולהגיב to סביבתה. כדי להעריך את תפקודו של חלבון קרום באופן מלא, ברור שהכרחי לדעת איך חלבון שמכוון ביחס לקרום שבתוכה הוא שוכן, כלומר, טופולוגיה הממברנה שלו. בנוסף לרכישת ידע מדעי בסיסי, הבנת הטופולוגיה של חלבון קרום ואילו היבטים של משטח חלבון נחשפים לסביבות שונות סימנה השלכות קליניות מאז חלבוני קרום מהווים את רוב המטרות תרופתיות 3. עד לאחרונה, גישות לקביעת טופולוגיה חלבון קרום דורשת השקעה רבה בזמן ובכסף, או שנדרש ריאגנטים שקשה להשיג. שני גישות ניסיוניות ובסיליקון ולהיות מועסקים על מנת לקבוע את הטופולוגיה של חלבוני הקרום המתגוררים בתוך קרום הפלזמה. מאז התחזיות הראשונות של תחומים קרום פורש המבוססים על ההערכה של הידרופוביות indiviחומצות אמינו כפולים 3, אלגוריתמים חזוי רבים זמינות כעת באינטרנט, ופשוט דורשות ידע של רצף החומצות האמיניות של החלבון. עם זאת, הנחות הן לעתים קרובות מרכזיות לתוכניות כגון דוגמנות, הנחות שיכולים להוביל למשימות לא נכונות של טופולוגיה 4,5. יתר על כן, בעוד שתחזיות מבוססות מחשב אלה יכולים בהיסוס להקצות אזורים פורש קרום, הם לא תמיד לקבוע אם אמינו או termini carboxy של חלבונים הם בציטופלסמה, לומן אברון או חיצוני תא. אפילו עם עליית כוח חישוב, והשימוש באלגוריתמי למידת מכונה 6, נתונים כאלה הוא עדיין מודל, וחייב להיות מאומת באמצעות נתונים שנרכשו באופן ניסיוני. קביעה ניסיונית ישירה של טופולוגיה קרום כבר התחייבה באמצעות פנלים של נוגדנים חד-שבטיים עם epitopes הידועה מופצת ברחבי החלבון, שבו הערכה של immunoreactivity נעשתה לפני ואחרי permeabilization התא. זהגישה דורשת מערך של נוגדנים, אשר לא יכול להיות זמינה לחלבון של עניין. אסטרטגיה חלופית היא להנדס תגי epitope כגון myc או hemagglutinin (HA) למקומות שונים ברחבי החלבון, ואחרי שוב על ידי קביעת immunoreactivity לפני ואחרי permeabilization הקרום. בנוסף לתגים חיסוניים, תגים האנזימטית (כולל phosphatase אלקליין, β -galactosidase, או -lactamase β) ושינויים כימיים כגון סריקת ציסטאין שכל הועסקו כדי לקבוע טופולוגיה חלבון קרום 7,8. שיטה נוספת המועסקות למיפוי טופולוגי של חלבוני קרום פלזמה מסתמכת על גישה מעט שונה לתגי epitope. בשיטה זו את רצף התג הוא רצף קונסנסוס glycosylation N הצמוד NXS / T. מאז glycosylation מתרחשת רק כאשר רצף זה נוכח בלומן של מסלול biosynthetic, הנוכחות של התג בluminal לעומתתא cytosolic הוא ציין בקלות כמו שינוי מסה על ג&#39;לי SDS-PAGE. גישה כזו הוחל ערוץ יון multispanning 9 CFTR. בעוד שכל הגישות הללו כבר נוצלו, ברור שכל מה שהם דורשים השקעות רבות בביולוגיה מולקולרית כדי ליצור רצף ומבני הסעפת. כדי לקבוע מידע טופולוגי לגבי חלבונים הטרנסממברני ממוקמים בתוך אברונים תוך-תאיים הוכיח להיות מאתגרים יותר. היישום של טכנולוגיות מבוססות הקרינה עם זאת, הפך את הנחישות של טופולוגיה קרום הרבה יותר פשוט. הטכניקה של שלמת הקרינה bimolecular (BiFC) מסתמכת על האינטראקציה בין שני שברים שאינם ניאון של חלבון פלואורסצנטי, שחזור מאפייני הניאון של החלבון ש-10. למרות שתחילה תאר לקביעת אינטראקציות בין חלבונים in vivo 10, גישה זו גם נוצלהכדי לקבוע טופולוגיה חלבון קרום בתאי צמח 11. עם זאת גישה זו היא גם זמן אינטנסיבי כפי שהוא דורש לא רק הדור של חלבוני היתוך עם החלבון של עניין, אלא גם מגוון של חלבוני היתוך ממוקדים ללום cytosol או אברון, ודורש ידע שבתאי קרומי החלבון של העניין מתגורר ב . גישה חלופית פשוטה יותר לקביעת טופולוגיה חלבון הקרום תוארה 12. Assay, הקרינה פרוטאז ההגנה (FPP) דורשת את הדור של חלבון היתוך בין GFP והגן של עניין. הגישה מבוססת על הנגישות היחסית של פרוטאזות שאינה ספציפית למחצית GFP; תלוי אם GFP מוגן מproteolysis על ידי המתגורר בלומן של אברונים תוך-תאיים או חשוף לproteolysis מעצם נוכחותו בcytosol. לכן, אם מחצית GFP על החלבון של עניין בפני ציטופלסמה, היא תהיה חשופה לפעילות פרוטאז ואותות הקרינה לאיבוד. לעומת זאת, אם מחצית GFP על החלבון של עניין בפני סביבה &#39;מוגנת&#39; מפרוטאז (כגון לום Golgi) ולאחר מכן את אות הקרינה תתמיד. כדי לאפשר פרוטאזות להיכנס לתא, אך לא להיכנס לתאי קרום תאיים digitonin תרופת הכולסטרול מחייב משמש. כולסטרול הוא סטרולים הדומיננטיים בבעלי חוליות, והוא מועשר באופן ספציפי בקרום הפלזמה ביחס לתאים תאיים. רעלן glycoside, digitonin, מופק אצבעונית הארגמנית צמח. יש digitonin זיקה לממברנות עשירות בכולסטרול, שבו היא מובילה לקרום בררני permeabiliztion 14,16 (איור 1). בנוסף מאפשר יציאה של רכיבי cytosolic קטנים, permeabilization digitonin גם מאפשר הכניסה של מולקולות חיצוניות, כגון K proteinase או טריפסין. פרוטוקול FPP מנצל את fact שקרום הפלזמה מכיל עד 80% מכולסטרול סלולארי 17, ואילו אברונים אחרים, כגון ER, Golgi, endosomes, מיטוכונדריה, שיש להם תוכן כולסטרול נמוך מאוד, נותרה על כנן 14. ההתאגדות סלקטיבית של כולסטרול לתוך קרום הפלזמה נצפתה בתאים רבים אוקריוטים, המתירה את השימוש בpermeabilization קרום הפלזמה digitonin תלוי במינים האיקריוטים מגוונים כגון S. cerevisiae ל14,18 אנושיים. Assay FPP מספק דרך פשוטה, מהירה וחזקה למדי של קביעה (א) אם חלבון הוא קרום מחויב / קשורים או diffusible בחופשיות בcytosol ו- (ב) שהתחום של חלבון קרום פונה ללום cytosol או אברון. צריך חלבון קרום אוריינטציות מרובות, האות תנבע מהצורה הדומיננטית וצורות קטין לא תזוהה. אמנם יש יכול להיות קצת חששו כי התוספת של מחצית GFP לחלבון על ריבית עשויה להשפיע על תפקודה ו/ או לוקליזציה subcellular, זה בעצם יותר תיאורטי מאשר בפועל. ואכן מחקרים רבים הראו באופן ברור כי תג ה- GFP לא לשנות את המאפיינים של החלבון 19,20.

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	<tbody>
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			<td height="20" style="height:20px;width:111px;">Name</td>
			<td style="width:80px;">Company</td>
			<td style="width:116px;">Catalgue Number</td>
			<td style="width:64px;">Comments</td>
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			<td height="20" style="height:20px;">Digitonin</td>
			<td>Calbiochem</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma</td>
			<td>T3924</td>
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			<td height="20" style="height:20px;">Cav1-GFP</td>
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			<td height="20" style="height:20px;">Polylysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Mounting Media</td>
			<td>Dako</td>
			<td>cS704</td>
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determining membrane protein topology using fluorescence protease protection (fpp)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

פלזמה ממברנה permeabilization permeabilization קרום פלזמה יעיל נקבע על ידי השימוש בחלבונים מסיסים ניאון (למשל, GFP, DsRed) (שלב 4). חלבונים אלה, כאשר באו לידי ביטוי בתאים, חופשיים מפוזר בcytosol, והולכים לאיבוד כאשר קרום פלזמת permeabilized באמצעות digiton...

Watch this Scientific Journal Video about Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP at JoVE.com

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

קביעת טופולוגיה חלבון ממברנה באמצעות הקרינה הגנת פרוטאז (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

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Biology

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

17.6K Views.  Chicago Medical School. Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP. 

Video: Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

שימוש בערכת איתור פלורסנט פרוטאז לקבוע פעילות פרוטאז

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

גיבוש חלבונים בממברנה עבור קביעת מבנה באמצעות Mesophases Lipidic

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

באמצעות מערכת GELFREE 8100 ההיפוך חלוקה על ההיפוך חלוקה משקל מבוסס מולקולרית עם שלב החילוץ נוזלי

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolecular השלמה פלואורסצנטי (BiFC) Assay עבור אינטראקציה בין חלבונים בתאים בצל באמצעות אקדח הליוס ג&#39;ין

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Gramicidin מבוססי Assay פלואורסצנטי; לקביעת מולקולות קטנות פוטנציאל שינוי מאפייני bilayer ליפידים

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

איתור של אינטראקציות חלבון הצמח באמצעות bimolecular תואם Gateway הקרינה השלמה (BiFC) מערכת

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

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

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

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

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

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

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

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

לוציפראז קומפלמנטציה הדמיה Assay העלים טבק benthamiana עבור Transiently קביעת חלבון אינטראקציה דינמיקה

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.