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.

التوجه الصحيح وطوبولوجيا من بروتينات الغشاء أمر ضروري لوظيفتها المناسبة. وعلى الرغم من أهمية فهم بروتين الغشاء طوبولوجيا، هناك العديد من البروتينات التي مثل هذه البيانات غير موجودة تماما. يوفر FPP طريقة سهلة وفعالة لتحديد بروتين الغشاء طوبولوجيا، واحد التي يمكن الق?...

أغشية البلازما، فضلا عن العديد من الأغشية داخل الخلايا، بمثابة الحواجز التي تفصل اثنين من مقصورات المائية. في حالة غشاء البلازما، والفصل هو بين الخارج والداخل من الخلية؛ لعضيات داخل الخلايا ومن بين السيتوبلازم والتجويف عضية. على سبيل المثال، غشاء الشبكة الإندوبلازمية (ER) يفصل بيئة المؤكسدة داخل تجويف ER من عصاري خلوي خفض البيئة 1. ويتم تخليق البروتينات الغشاء على ريبوسوم المرتبط ER، وتحقيق طوبولوجيا النهائي داخل غشاء ER 2. الاستيلاء على التوجه غشاء المناسب وطوبولوجيا للبروتينات هو أمر حاسم لوظيفتها الطبيعية. طوبولوجيا الصحيح يسمح المجالات ذات الصلة للبروتينات غشاء للتفاعل مع الشركاء ملزمة لها، لأنها تتيح التعديلات بعد متعدية حاسمة لتحدث، وذلك في حالة من البروتينات غشاء البلازما، بما يسمح للخلية للتفاعل مع ويستجيب رس بيئته. لنقدر تماما وظيفة بروتين الغشاء، لا بد من الواضح أن تعرف كيف يتم توجيه هذا البروتين فيما يتعلق غشاء من خلاله أنه يقيم، أي طوبولوجيا غشاء لها. بالإضافة إلى اكتساب المعرفة العلمية الأساسية، فهم طوبولوجيا من بروتين الغشاء والتي يتعرض لها جوانب سطح البروتين لبيئات مختلفة ميزت الآثار السريرية منذ تتكون البروتينات الغشاء غالبية أهداف الدوائية 3. حتى وقت قريب، يقترب إلى تحديد بروتين الغشاء طوبولوجيا كان يتطلب استثمارات كبيرة في الوقت والمال أو كان يتطلب الكواشف التي يصعب الحصول عليها. على حد سواء قد استخدمت النهج التجريبية وفي سيليكون لتحديد طوبولوجيا غشاء البروتينات المقيمين داخل غشاء البلازما. منذ التنبؤات الأولى من المجالات الغشاء الذي يمتد على أساس للا مائية تقييمها من indiviالأحماض الأمينية المزدوجة 3، والعديد من خوارزميات التنبؤ متوفرة الآن على شبكة الإنترنت، وببساطة تتطلب معرفة تسلسل الأحماض الأمينية والبروتين. ومع ذلك، غالبا ما تكون الافتراضات المركزية لمثل هذه البرامج النمذجة، والافتراضات التي يمكن أن تؤدي إلى مهام غير صحيحة طوبولوجيا 4،5. وعلاوة على ذلك، في حين يمكن لهذه التنبؤات القائمة على الحاسوب بتعيين مبدئيا المناطق التي تغطي الغشاء، أنها لا تحدد دائما ما إذا كان الأمينية أو مصطلحات كربوكسي البروتينات هي في السيتوبلازم، التجويف عضية أو الخارجي الخلية. حتى مع زيادة القوة الحسابية، واستخدام آلة التعلم خوارزميات 6، مثل هذه البيانات لا تزال نموذجا، ويجب التحقق من صحة باستخدام البيانات التي حصل عليها تجريبيا. تم إجراء تقرير التجريبي المباشر للطوبولوجيا الغشاء باستخدام لوحات من الأجسام المضادة وحيدة النسيلة مع الحواتم المعروف توزع في جميع أنحاء البروتين، حيث أحرز تقييم مناعية، قبل وبعد permeabilization الخلية. هذاالنهج يتطلب مجموعة من الأجسام المضادة، التي قد لا تكون متاحة للبروتين من الفائدة. استراتيجية بديلة هو مهندس علامات حاتمة مثل MYC أو هيماغلوتينين (HA) في مواقع مختلفة في جميع أنحاء البروتين، ثم مرة أخرى عن طريق تحديد مناعية قبل وبعد permeabilization الغشاء. بالإضافة إلى علامات المناعية، كلها قد استخدمت علامات الأنزيمية (بما في ذلك الفوسفاتيز القلوية، β -galactosidase، أو β -lactamase) والتعديلات الكيميائية مثل مسح السيستين لتحديد بروتين الغشاء طوبولوجيا 7،8. طريقة إضافية تستخدم لرسم الخرائط الطوبوغرافية للبروتينات غشاء البلازما يعتمد على مقاربة مختلفة قليلا إلى علامات حاتمة. في هذه الطريقة تسلسل العلامة هو N-ربط تسلسل إجماع بالغليكوزيل NXS / T. منذ بالغليكوزيل يحدث فقط عندما مثل هذا التسلسل هو موجود في تجويف مسار السكروز، فإن وجود علامة في اللمعية مقابلويلاحظ حجرة عصاري خلوي بسهولة كما تحول الشامل على المواد الهلامية SDS-PAGE. وقد تم تطبيق هذا النهج إلى multispanning القناة الايونية CFTR 9. في حين استخدمت كل هذه الأساليب، فمن الواضح أنهم جميعا تتطلب استثمارات كبيرة في مجال البيولوجيا الجزيئية لتوليد وتسلسل بنيات متعددة. لتحديد المعلومات الطوبوغرافية المتعلقة البروتينات عبر الغشاء الموجود داخل عضيات داخل الخلايا وقد ثبت أن أكثر تحديا. تطبيق التكنولوجيات القائمة مضان ومع ذلك، جعلت تحديد الغشاء طوبولوجيا أبسط كثيرا. تقنية ثنائي الجزيء تكامل مضان (BiFC) تعتمد على التفاعل بين اثنين من شظايا غير الفلورية من بروتين فلوري، واستعادة خصائص الفلورسنت من أن البروتين 10. على الرغم من وصف في البداية لتحديد تفاعلات البروتين البروتين في الجسم الحي 10، كما تم استخدام هذا النهجلتحديد بروتين الغشاء طوبولوجيا في الخلايا النباتية 11. ولكن هذا النهج هو أيضا الوقت المكثف لأنه يتطلب الجيل ليس فقط من البروتينات الانصهار مع بروتين من الفائدة ولكن أيضا مجموعة متنوعة من البروتينات الانصهار تستهدف العصارة الخلوية أو عضية التجويف، ويتطلب المعرفة التي بين الخلايا الأغشية البروتين من الفائدة تكمن في . وقد وصفت نهج بديل أبسط لتحديد بروتين الغشاء طوبولوجيا 12. الفحص، الإسفار البروتياز الحماية (FPP) يتطلب توليد البروتين الانصهار بين GFP والجينات في المصالح. ويستند هذا النهج على إمكانية الوصول النسبي للالبروتياز غير محددة لشاردة GFP. اعتمادا على ما إذا GFP محمي من التحلل البروتيني من قبل المقيمين في لمعة من العضيات داخل الخلايا أو يتعرض لالتحلل البروتيني من خلال كونها موجودة في العصارة الخلوية. وهكذا، إذا شاردة GFP على البروتين من الفائدة تواجه السيتوبلازم، فانها ستكون معرضة لنشاط الأنزيم البروتيني وإشارة مضان خسر. على العكس، إذا شاردة GFP على البروتين من الفائدة تواجه بيئة &#39;محمية&#39; من البروتيني (مثل التجويف جولجي) ثم فإن إشارة مضان تستمر. للسماح البروتياز لدخول الخلية، ولكن لا تدخل مقصورات غشاء الخلايا ويستخدم الكوليسترول ملزمة ديجيتونين المخدرات. الكولسترول هو ستيرول المهيمن في الفقاريات، وإثراء تحديدا في غشاء البلازما نسبة إلى المقصورات داخل الخلايا. السم غليكوزيدات، ديجيتونين، يستخرج من نبات الديجيتال الأرجواني. ديجيتونين يتعاطف مع الكولسترول أغشية الغنية، حيث أنه يؤدي إلى غشاء انتقائي permeabiliztion 14،16 (الشكل 1). بالإضافة إلى السماح بخروج عناصر عصاري خلوي صغيرة، يسمح permeabilization ديجيتونين أيضا دخول جزيئات الخارجية، مثل K بروتين أو التربسين. بروتوكول FPP يستفيد من القوات المسلحة الكونغوليةر التي تحتوي على غشاء البلازما تصل إلى 80٪ من الكولسترول الخلوي 17، في حين العضيات الأخرى، مثل ER، جولجي، الإندوسومات، الميتوكوندريا، والتي تحتوي على محتوى الكولسترول منخفض جدا، لا تزال سليمة 14. وقد لوحظ إدراج الانتقائي للالكولسترول في غشاء البلازما في العديد من الخلايا حقيقية النواة، السماح باستخدام تعتمد على ديجيتونين permeabilization غشاء البلازما في مثل هذه الأنواع حقيقية النواة متنوعة مثل S. الخباز لالبشري 14،18. يوفر الفحص FPP وسيلة بسيطة وسريعة وقوية نسبيا من تحديد (أ) ما إذا كان البروتين هو غشاء ملزمة / اسوشيتد أو إنتشاري بحرية في العصارة الخلوية و (ب) والذي المجال من البروتين غشاء يواجه العصارة الخلوية أو عضية التجويف. يجب أن بروتين الغشاء لديها توجهات متعددة، فإن إشارة تنشأ من النموذج المهيمن، ولن يتم الكشف عن أشكال طفيفة. في حين يمكن أن يكون هناك بعض القلق من أن إضافة شاردة GFP للبروتين على الفائدة قد تؤثر على وظيفة، و/ أو توطين التحت خلوية، وهذا هو في الواقع أكثر من النظري الفعلية. في الواقع العديد من الدراسات قد أظهرت بوضوح أن العلامة GFP لا يغير خصائص البروتين 19،20.

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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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			<td height="20" style="height:20px;">Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma</td>
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			<td height="20" style="height:20px;">Cav1-GFP</td>
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			<td>Clontech</td>
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			<td height="20" style="height:20px;">Polylysine</td>
			<td>Sigma</td>
			<td>P4707</td>
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			<td height="20" style="height:20px;">Mounting Media</td>
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			<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.

البلازما غشاء Permeabilization يتم تحديد كفاءة permeabilization غشاء البلازما عن طريق استخدام البروتينات الفلورية القابلة للذوبان (على سبيل المثال، GFP، DsRed) (الخطوة 4). هذه البروتينات، عندما أعرب في الخلايا، أحرار في نشر في العصارة الخل...

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.

تحديد غشاء طوبولوجيا البروتين عن طريق الإسفار البروتياز الحماية (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.

استخدام أدوات الكشف البروتياز نيون تحديد نشاط الأنزيم البروتيني

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.

بلورة البروتينات الغشائية لتحديد الهيكل باستخدام 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.

باستخدام نظام التقطير GELFREE 8100 لتقطير الوزن الجزيئي وبناء مع مرحلة الانتعاش السائل

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)

ثنائي الجزيء الإسفار التكملة (BiFC) الفحص عن البروتين البروتين في خلايا البصل التفاعل باستخدام مدفع الجينات هيليوس

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.

غراميسيدين القائم على الفحص الإسفار ؛ لتحديد الجزيئات الصغيرة المحتملة لتعديل خصائص طبقة ثنائية الدهن

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.

الكشف عن تفاعلات البروتين في النبات باستخدام عبارة الإسفار ثنائي الجزيء المتوافقة التكملة (BiFC) نظام

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.

في دراسة لالإلتقام وإعادة غشاء البلازما من البروتينات القائم على خلية L-الجلوتاثيون حماية فحوصات

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.

الأخضر الفلورسنت القائم على البروتين التعبير فحص غشاء البروتينات في<em&gt; الإشريكية القولونية</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.

لوسيفراس "التكامل بالتصوير الإنزيم" في أوراق نيكوتيانا بينثاميانا "تحديد عابر ديناميات التفاعل" البروتين-بروتين

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.