وأيد هذا العمل من خلال منح المعاهد الوطنية للصحة وR01NS046835 R01AG034389 ، ومارس من منحة الدايمات 6FY08 - 322. شكر خاص مكرس لك السيدة اليانور براون (Miltenyi بيوتيك ، صحراء ، كاليفورنيا) الذي ساعد بشكل كبير على بصيرة لها في MACS التكنولوجيا. وقد قدمت بسخاء Miltenyi المواد المستخدمة للمظاهرة من التجارب في أي تكلفة. كما أنني ممتن للدكتور جوانجهو وانغ (كلية الطب في جورجيا / جامعة جورجيا العلوم الصحية ، أوغوستا ، GA) الذي ولدت PKCζ معربا عن خطوط الخلايا. ومن المسلم به أيضا دعم من قبل معهد الطب الجزيئي في كلية الطب في جورجيا / جامعة جورجيا للعلوم الصحية (في إطار الاتجاه من الدكتور لين مى).

1. مقدمة تم تطوير اللوني الدهون تقارب حويصلة بوساطة استخدام الفرز المغناطيسي تنشيط الخلية (LIMACS) تقنية في المختبر لدينا لعزل بروتين المجمعات سيراميد المرتبطة 1-3. في الأصل ، أدلى الحويصلات الدهني سيراميد وفسفاتيديل ، مما سمح لMACS باستخدام الجسيمات المغناطيسية مترافق Annexin الخامس (أفيني للغاية لفسفاتيديل) لعزل الحويصلات والبروتينات المرتبطة بها. وقد استخدمنا أسلوب LIMACS لإعادة المختبر في مجمع الاقطاب سيراميد المرتبطة وعزل سيراميد ملزم البروتينات من الخلية lysates 3. يمكن تعديل LIMACS به الشركاء التفاعل أخرى لعزل الحويصلات (على سبيل المثال ، شحمي سكري ، أو الأجسام المضادة specifc lectins الدهن). 2. الإجراءات التجريبية إعداد الحويصلات الدهن وفحوصات aPKCBinding <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويتم الحصول على حويصلات الدهنية من الخلائط المجففة من المبالغ متساوي المولية للفسفاتيديل (105 ميكروغرام) و C16 - سيراميد (85 ميكروغرام) وفقا للإجراءات معدلة لإعداد الحويصلية 1،4-7 كبيرة. </li><li style=";text-align:right;direction:rtl"> ومعلق ثم خليط الدهن وsonicated ل 1 ح في 100 ميكرولتر من العازلة حويصلة تتكون من تريس 50 مم / حمض الهيدروكلوريك (الرقم الهيدروجيني 7.5) و 150 ملي مول كلوريد الصوديوم. </li><li style=";text-align:right;direction:rtl"> بعد إضافة 300 ميكرولتر من حويصلة العازلة تستكمل مع MnCl 0.1 ملم 2 ، يتم طرد هذه العينات في ز μ 12000 لمدة 20 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> هو معلق على بيليه (حويصلات الدهنية الكبيرة) في 100 ميكرولتر من حويصلة العازلة وحضنت مع 1 nmol من Vybrant الجاذبة - CM : 1 ساعة عند 37 درجة مئوية لتصور جزء الحويصلة بعد الانفصال MACS. Vybrant CM - الجاذبة هو صبغ أحمر فلوري دمج تحديدا في الأغشية الدهنية. </li><li style=";text-align:right;direction:rtl"> ويتم إعداد المنظفات الخالية من lysate الخلية صوتنة / التجانس من الخلايا في 300 ميكرولتر من ناقص التوتر العازلة (10 ملي تريس / حمض الهيدروكلوريك (7.0 درجة الحموضة) ومثبطات الأنزيم البروتيني مع الفوسفاتيز) ، يليه إزالة الحطام الغشائي بواسطة الطرد المركزي. وينبغي أن يضاف خطوة الطرد المركزي في 100000 XG ل 1 ح لتجنب تلوث lysate الخلية أغشية داخلية تحتوي على فسفاتيديل. </li><li style=";text-align:right;direction:rtl"> إضافة إلى مسح lysate الدهون تعليق الحويصلة ، وحضنت الخليط لمدة 2 ساعة عند 4 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> ثم تستكمل خليط التفاعل مع 20 ميكرولتر من 20X العازلة الخامس Annexin ملزم و 50 ميكرولتر من محلول يحتوي على حبات المغناطيسي مترافق إلى الخامس Annexin بواسطة الحضانة لمدة 1 ساعة عند 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> يتم تنفيذ MACS وفقا لالصانع (Miltenyi بيوتيك ، وشركة) البروتوكول. يتم تحديد وجود وكمية من الدهون الحويصلات عن طريق رصد Vybrant CM - الجاذبة مضان في التدفق من خلال كسور وشطف باستخدام قارئ microplate مضان. </li><li style=";text-align:right;direction:rtl"> العلاقة الخطية بين كمية الدهون الحويصلي وكثافة مضان الحويصلة متجهة Vybrant CM - الجاذبة يتم التحقق من الكمية التي اللوني رقيقة الطبقة العالية الأداء (HPTLC) من خليط الدهن تطبيقها على أساس MACS V - Annexin. </li><li style=";text-align:right;direction:rtl"> يتم التحقق من نوعية رد الفعل ملزم للبروتينات aPKC أو غيرها إلى الحويصلات سيراميد / فسفاتيديل قبل مقايسة الأضداد المنافسة باستخدام 1 ميكروغرام المضادة للأجسام المضادة PKCζ الأرنب النسائل المتعددة لاحتضان هذه الخلية لlysate ح 1 في 4 درجات مئوية قبل الحضانة مع وحويصلات الدهنية. </li><li style=";text-align:right;direction:rtl"> ويتم تحليل هذا البروتين ملزمة لسيراميد / فسفاتيديل الحويصلات في شطافة MACS بواسطة SDS - PAGE وimmunoblotting. </li></ol> في المختبر معقدة البروتين الدهني قطبية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم تنفيذ إعادة المختبر في مجمع الاقطاب البروتين الدهني باتباع الإجراء LIMACS كما هو موضح في المقطع السابق. في فسفاتيديل وجيزة (420 ميكروغرام) و C16 - سيراميد (107 ميكروغرام) يجفف من المذيبات العضوية. </li><li style=";text-align:right;direction:rtl"> ومعلق في الدهون تحت صوتنة المجفف في 500 ميكرولتر من حويصلة العازلة (50 ملي تريس / حمض الهيدروكلوريك ، ودرجة الحموضة 7.5 ؛ 150 مم كلوريد الصوديوم). </li><li style=";text-align:right;direction:rtl"> يضاف خمسة ميكرولتر من 10 ملي MnCl2 ، 1 ميكرولتر من Vybrant الجاذبة - CM ، و 500 نانوغرام من PKCζ (المؤتلف الإنسان) وخليط التفاعل المحتضنة undergentle الانفعالات لمدة 60 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> يتم استرداد CM - الجاذبة ملطخة Vybrant فسفاتيديل / سيراميد حويصلات بواسطة الطرد المركزي في 12000 XG لمدة 60 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> هو معلق على بيليه (الوردي) في 100 تريس العازلة ميكرولتر وتستكمل مع γS - GTP (100 ميكرومتر) ، الناتج المحلي الإجمالي (1 ملم) ، وضريبة السلع والخدمات ، Par6 (100 نانوغرام) ، أو ضريبة السلع والخدمات ، Cdc42 (500 نانوغرام) وكذلك حضنت لمدة 3 ح في 4 درجات مئوية (يمكن استخدام أي تركيبة أخرى من البروتينات المؤتلف من الاهتمام هنا). </li><li style=";text-align:right;direction:rtl"> Annexin V - العازلة (5 ميكرولتر من محلول المخزون 20X) ، وAnnexin V - مترافق الخرز المغناطيسي (50 ميكرولتر) وتضاف إلى خليط التفاعل في إطار التحريض المحتضنة لطيف لمدة 30 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> يتم تنفيذ Annexin V - MACS بعد بروتوكول المورد كما هو موضح سابقا. </li&gt; <li style=";text-align:right;direction:rtl"> وتستكمل الكسر شطف (1 مل) مع 10 ميكروغرام من ovalbumin نقية كمعونة هطول الأمطار. ويتركز هذا البروتين التي يسيل - Flugge هطول الأمطار وتحليلها بواسطة SDS-PAGE/immunoblotting كما هو موضح سابقا 8. </li><li style=";text-align:right;direction:rtl"> كميا هو كمية الدهون eluted الحويصلات التي كشف Vybrant الجاذبة - CM (الوردي) في المرحلة (كلوروفورم / الميثانول) العضوي للتفاعل Flugge الأمطار يسيل. هو تطبيع كمية البروتين في تحليل كميات متساوية من حويصلات الدهنية. </li></ol> 3. النتائج LIMACS تنقية EGFP - PKCζ والمجال سيراميد ملزمة C20ζ - EGFP A lysate المنظفات الخالية من الخلايا MDCK معربا عن كامل طول PKCζ C - عضال مرتبطة بروتين الفلورية الخضراء (FLζ - EGFP) أو مجال سيراميد ملزمة في النهاية من PKCζ - C (EGFP - C20ζ) وحضنت مع فسفاتيديل / الحويصلات كما سيراميد ووصف في الإجراءات التجريبية. بعد شطف من العمود MACS ، تم تحليل البروتينات والأجسام المضادة باستخدام immunoblotting ضد PKCζ وEGFP للكشف عن البروتين eluted 2. <img src="/files/ftp_upload/2657/2657fig1.jpg" alt="الشكل 1" /> الشكل 1. LIMACS من EGFP المسمى PKCζ والخمسين من طراز C - المحطة باستخدام جزء C20ζ فسفاتيديل / سيراميد الحويصلات. وحضنت المنظفات الخالية من الخلايا lysates MDCK معربا عن EGFP (كعنصر تحكم غير ملزمة) ، كامل طول PKCζ - EGFP ، أو الربط سيراميد ، C - المحطة جزء C20ζ - EGFP مع فسفاتيديل / حويصلات سيراميد كما هو موضح في المقطع الإجراءات التجريبية . بعد استخدام LIMACS ، كان eluted البروتين العازلة مع عينة وتحليلها بواسطة SDS SDS - PAGE وimmunoblotting. اللوحة اليسرى يدل على أن لا ربط EGFP إلى الحويصلات مع الاحتفاظ حبات V - مرتبطة Annexin المغناطيسي. لوحة المتوسطة وتبين أن الحق الكامل طول PKCζ - EGFP واستبقيت C20ζ - EGFP بسبب ملزمة لسيراميد.

<ol>
	<li>Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+to+ceramide+activates+protein+kinase+Czeta+before+the+formation+of+a+pro-apoptotic+complex+with+PAR-4+in+differentiating+stem+cells.">Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.</a> J Biol Chem. 280, 26415-26424 (2005).</li><li>Wang, G., Krishnamurthy, K., Umapathy, N. S., Verin, A. D., Bieberich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carboxyl-terminal+domain+of+atypical+protein+kinase+Czeta+binds+to+ceramide+and+regulates+junction+formation+in+epithelial+cells.">The carboxyl-terminal domain of atypical protein kinase Czeta binds to ceramide and regulates junction formation in epithelial cells.</a> J Biol Chem. 284, 14469-14475 (2008).</li><li>Chalfant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+ceramide+regulates+the+alternative+splicing+of+caspase+9+and+Bcl-x+in+A549+lung+adenocarcinoma+cells.+Dependence+on+protein+phosphatase-1.">De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.</a> J Biol Chem. 277, 12587-12595 (2002).</li><li>Simon, C. G., Holloway, P. W., Gear, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+C(16)-ceramide+between+phospholipid+vesicles.">Exchange of C(16)-ceramide between phospholipid vesicles.</a> Biochemistry. 38, 14676-14682 (1999).</li><li>Goni, F. M., Contreras, F. X., Montes, L. R., Sot, J., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+(and+sociology)+of+ceramides.">Biophysics (and sociology) of ceramides.</a> Biochem Soc Symp. , 177-188 (2005).</li><li>Kumagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CERT+mediates+intermembrane+transfer+of+various+molecular+species+of+ceramides.">CERT mediates intermembrane transfer of various molecular species of ceramides.</a> J Biol Chem. 280, 6488-6495 (2005).</li><li>Wessel, D., Flugge, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+quantitative+recovery+of+protein+in+dilute+solution+in+the+presence+of+detergents+and+lipids.">A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.</a> Anal Biochem. 138, 141-143 (1984).</li></ol>

الإعلان عن أي تضارب في المصالح.

لاختبار مدى التفاعل بين محددة الدهون والبروتين ملزمة تعوقها تضمين الدهون في غشاء الخلية. غشاء الخلية يتكون من خليط من عدة الدهون والبروتينات ويتم تنظيمه في microdomains الدهون أو الطوافات. لذلك ، يمكن المشارك تنقية microdomains والبروتينات لا يميز بوضوح ما إذا كان بروتين يرتبط ...

وقدمت Annexin V - مترافق المغناطيسي والخرز الصغير وMACS minicolumns من قبل شركة Miltenyi ، بيوتيك (صحراء ، CA). كانت كل الدهون من أعلى نقاء والحصول عليها من الدهون أفانتي القطبية (المرمر ، AL).

lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction

تحليل التفاعل البروتين الدهني صعب جدا ، لأن جزءا لا يتجزأ من الدهون في أغشية الخلايا ، وبالتالي ، لا يمكن الوصول إليها إلى إجراءات أشد تنقية. كبديل ، يمكن أن تكون مغلفة الدهون على الأسطح المسطحة كما تستخدم لمأكل مثل الطحين والدهن ELISA الطيفي بالرنين. ومع ذلك ، والدهون طلاء السطح لا تشكل هياكل microdomain ، والتي قد تكون مهمة لخصائص الربط الدهون. مزيد من هذه الطرق لا تسمح لتنقية كميات أكبر من البروتينات ملزمة الدهون المستهدفة. للتغلب على هذه القيود المفروضة على اختبار البروتين الدهني التفاعل وتنقية البروتينات الشحمية ملزمة طورنا طريقة جديدة تسمى اللوني الدهون تقارب حويصلة بوساطة استخدام الفرز المغناطيسي تنشيط الخلية (LIMACS). في هذه الطريقة ، يتم إعداد حويصلات الدهن مع وجود الهدف والدهون مثل الدهون فسفاتيديل مرساة لMACS Annexin الخامس. فسفاتيديل هو غشاء الخلية التي تظهر في كل مكان فسفوليبيد تقارب عالية لبروتين خامسا Annexin باستخدام الخرز المغناطيسي مترافق إلى الخامس Annexin الحويصلات التي تحتوي على الدهون فسفاتيديل سوف ربط الخرز المغناطيسي. متى يتم تحضين الحويصلات الدهون مع خلية lysate أيضا البروتين الدهني ملزما للهدف أن تكون ملزمة للالخرز ويمكن أن يشترك في تنقية MACS به. ويمكن أيضا أن تستخدم هذه الطريقة لمعرفة ما إذا البروتينات المؤتلف إعادة مجمع البروتين الدهني ملزما للهدف. وقد استخدمنا هذا الأسلوب لإظهار التفاعل بين PKC شاذة (aPKC) مع سيراميد الشحميات السفينغولية وشارك في تنقية استجابة الخلايا البروستاتا 4 (PAR - 4) ، وهو بروتين ملزمة لسيراميد المرتبطة aPKC. وقد استخدمنا هذا الأسلوب أيضا لإعادة هيكلة مجمع سيراميد المرتبطة من aPKC المؤتلف مع الخلية القطبية ذات الصلة والبروتينات Par6 Cdc42. حيث يمكن إعداد حويصلات الدهنية مع مجموعة متنوعة من الدهون الفوسفاتية أو sphingo ، LIMACS يقدم تنوعا لاختبار البروتين الدهني التفاعل في بيئة الدهون التي تمثل بشكل وثيق من أن غشاء الخلية. يمكن التعرف على البروتين الدهني إضافية المجمعات باستخدام تحليل البروتينات من البروتين الدهني الملزم الذي اشترك في المنقى مع حويصلات الدهنية.

Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

لاختبار مدى تفاعل البروتين الدهني مع هدفها كنا MACS وAnnexin V - مترافق الخرز المغناطيسي وحويصلات الدهنية من الدهون المركبة المستهدفة وAnnexin فسفاتيديل V - ملزمة. هي بروتينات الدهنية منضم إلى الهدف المشترك تنقيتها وتحليلها بعد شطف من الخرز.

الكروماتوجراف الدهون تقارب الحويصلة بوساطة الفرز المغناطيسي باستخدام خلية المنشط (LIMACS) : طريقة جديدة لتحليل البروتين الدهني التفاعل

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

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Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

12.4K Views.  Georgia Health Sciences University. لاختبار مدى تفاعل البروتين الدهني مع هدفها كنا MACS وAnnexin V - مترافق الخرز المغناطيسي وحويصلات الدهنية من الدهون المركبة المستهدفة وAnnexin فسفاتيديل V - ملزمة. هي بروتينات الدهنية منضم إلى الهدف المشترك تنقيتها وتحليلها بعد شطف من الخرز.

Video: Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

مضان الفرز الخلية النباتية المنشط من Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

عزل بروتين المجمعات المتعددة التي كتبها عطوب في الجسم الحي الشواهد الخلوية عبر ربط والمناعة المغناطيسي اللوني التقارب

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

الطمع القائم على فحص التفاعل خارج الخلية (AVEXIS) للكشف قابلة منخفضة تقارب التفاعلات مستقبلات يجند خارج الخلية

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

تقارب المرحلة السائلة الفحص التقاطها باستخدام الخرز المغناطيسي لدراسة البروتين البروتين التفاعل: المثال فيروس شلل الأطفال، Nanobody

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

تحليل خلية معينة من<em&gt; ل arabidopsis</em&gt; يترك طريق الإسفار خلية المنشط الفرز

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

الأمثل أساليب النمذجة وتلوين لرصد انتشار استخدام الأصباغ انقسام الخلية خلية تتبع

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

الجمع بين الفرز المغناطيسي للخلايا الأم والاختبارات تذبذب لتحليل الجينوم عدم الاستقرار خلال الإنقسامية شيخوخة الخلايا في<em&gt; خميرة الخباز</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

تنقية مجمعات الأصلية للدراسة الهيكلية باستخدام طريقة العلامة جنبا الى جنب الانجذاب

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

مغناطيسية تنشيط الخلية فرز الاستراتيجيات الرامية إلى عزل وتنقية الزليلي المستمدة من سائل الخلايا الجذعية الوسيطة من طراز الأرنب

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

أسلوب الرواية بلازميد الحمض النووي إيصالها إلى الماوس المثانة أوروثيليوم انهانسر

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...