أولا ذوبان للخلايا يأخذ قنينة TN5 المجمدة للخلايا ، وذوبان الجليد في حمام ماء C ° 37. إضافة 5ml المتوسطة عبر عن الحشرات (Invitrogen ، وأنواع ، عدد 10486) في دورق 25cm TC 2. علما بأن اكسبريس five يأتي دون الجلوتامين ، تحتاج إلى إضافة 10ML من بكتيريا القلم الجلوتامين 100X أو الجلوتامين وحدها إلى وسائل الإعلام ليتر 1. إضافة الى ذلك TN5 الخلايا واحتضان لمدة 30 دقيقة في الحاضنة 27 درجة مئوية. ثم نضح المتوسط ​​مع DMSO بلطف من دون إزعاج الخلايا التي تعلق على الجزء السفلي من القارورة. إضافة 5ml المتوسطة الطازجة. II. المتنامية والتوسع في خلايا رصد خلايا في كل يوم. عندما القارورة ما يقرب من 70 ٪ متموجة ، وجعل الخلايا في التعليق من ضجيجا القارورة على كلا الجانبين ، ونقل الخلايا إلى 175cm 2 قارورة بإضافة 21 مل من التعبير عن الحشرات والمتوسطة 5ml للثقافة الخلية. وينبغي أن يكون كل قارورة T175 حوالي 25 - 30ml المتوسط ​​كوحدة تخزين النهائي. انقسام قارورة متموجة (حوالي 1 مليون / مل اضرب.) 1 / 4 أو 1 / 5 حسب الحاجة. لا تدع الخلايا كسا الأرض. عد عدد من المقاطع التي لديك انقسام الخلايا. لا تنمو أكثر من عدد مرور 30 ​​لأنه قد يسبب شيخوخة الخلايا الناتجة تحلل الخلية. عندما تصل إلى حجم المطلوب في تركيز 1 مل / مليون تصيب الخلايا. أنا تنمو عادة ما يصل إلى 2 إلى 2.5 ليتر ، والذي يصل عادة إلى قوارير 175cm 2 وسبعين تقريبا. 25 إلى قارورة 30ml / أو خمسة مخروطي 1 لتر وقوارير تقريبا. 400ml الى 500 مل في كل قارورة ملاحظة : إما يمكن أن تنمو في خلايا T - 175 سد القارورة الختم أو كورنينج دورق مخروطي. الخلايا المتنامية في قوارير مخروطي هو أسرع وأسهل. III. Titering الفيروس عن طريق نقطة النهاية أسلوب التخفيف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لوحة 3000-5000 الخلايا بشكل جيد في شقة في لوحة أسفل بشكل جيد في 96 200ul المتوسطة اكسبريس SFM الخمسة. السماح للخلايا تستقر عند 27 درجة مئوية لمدة 30 دقيقة على الأقل. </li><li style=";text-align:right;direction:rtl"> جعل المسلسل التخفيف من الأسهم الفيروسة العصوية. ينبغي للمرء أن يفعل 10/01 التخفيف في سطر واحد من 96 لوحة أسفل U جيدا ، وذلك باستخدام 250ul لكل بئر. يتم إجراء التخفيفات في المتوسط ​​خمسة اكسبريس SFM. </li><li style=";text-align:right;direction:rtl"> باستخدام ماصة الأقنية ، ونقل مبلغ ثابت (عادة 20 ماي) من التخفيفات المختلفة للخلايا. </li><li style=";text-align:right;direction:rtl"> ثقافة حتى 27 درجة مئوية لمدة 7 أيام. لتجنب التبخر من حافة الصفوف ، والتفاف حواف الصفيحة مع parafilm. </li><li style=";text-align:right;direction:rtl"> بعد 7 أيام ، والتحقق من لوحة وتحديد التخفيف من فيروس العدوى يعطي 50 ٪ -- نصف عدد المصابين بهذا المرض في هذه الآبار تركيز الفيروس). ثم استخدام صيغة لحساب عيار ، وهو ما يعبر عنه في pfu / مل (pfu - البلاك تشكيل وحدات). تم العثور على أدلة في الصيغة الأكثر الفيروسة العصوية. </li></ol> رابعا. اصابة الخلايا من المهم جدا أن نعرف بالضبط من عيار الفيروس. إضافة المبلغ المطلوب من الفيروس. وينبغي لإعداد المخزون الفيروسية تصاب الخلايا في تعدد العدوى (وزارة الداخلية) لا تزيد عن 1 (1pfu 1High إلى خمسة الخلية) العالي وزارة الداخلية يؤدي إلى إنتاج الطفرات الفيروسية. لإنتاج البروتين ، والمبلغ المعتاد هو MO1 50-10. على سبيل المثال : وعيار من الفيروسة العصوية mCD1d = 1X108 pfu / مل وزارة الداخلية إضافة = 1 (لتضخيم الفيروس) = 20X10 ^ 6 خلية / قارورة / 1X10 ^ 8 pfu / مل UL = 200 / القارورة وزارة الداخلية = 5 إلى 10 (لإنتاج بروتين) = 1ml 2ml بالنسبة إلى 20x10 ^ 6 خلية / القارورة في يوم 4 بعد العدوى ، وننظر إلى الخلايا المصابة. يجب فصل فيه ، العائمة ، والنقانق وتشكيل تورم. خامسا استعادة Supernatants تتخذ وسيلة من قوارير المصابة ، ونقل إلى اللون الأزرق زجاجات 250ml فالكون غطاء للغزل. ويمكن تعقيمها أنهم وإعادة استخدامها. تعبئة الزجاجات بالتساوي والخلايا في جهاز الطرد المركزي Sorvall GSA الدوار في 200rpm ، 20 دقيقة ، 4 درجات مئوية. جمع طاف والمضي قدما نحو المزيد من التنقية. سادسا. تنقية المؤتلف mCD1d <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسيل الكلى وتركيز الصوديوم في 150mM العازلة الفوسفات (درجة الحموضة 7.4) </li><li style=";text-align:right;direction:rtl"> بواسطة الكروماتوجراف Agarose ني NTA ، أزل البروتين mCD1d + b2m مع 150mM الصوديوم Immidazole الفوسفات ملي +200 </li><li style=";text-align:right;direction:rtl"> تشغيل عمود التحلية باستخدام 20mM تريس هيدروكلورايد pH8 العازلة للتخلص من Immidazole </li><li style=";text-align:right;direction:rtl"> تشغيل Exchange باستخدام الكروماتوغرافيا الأنيون MonoQ العمود للقضاء على الملوثات المتبقية. </li><li style=";text-align:right;direction:rtl"> التركيز على استخدام 1mg/ml YM 30 المكثف (ميليبور) </li><li style=";text-align:right;direction:rtl"> mCD1d البيرا biotynlation الأنزيمية وفقا لبروتوكول من الشركة المصنعة (الطمع) </li><li style=";text-align:right;direction:rtl"> يتم التخلص من البيوتين S200 مجانا من العمود (حجم Chromatograpy الاستبعاد) باستخدام الفوسفات مخزنة المالحة (PBS) ، pH7.4. ويمكن الحصول على المتوسط ​​من 2 إلى 3 ملغ من البروتين biotynlated من 2 لتر من طاف </li><li style=";text-align:right;direction:rtl"> إنتاج - Galcer CD1d tetramer. </li></ol> من أجل تحميل جزيء CD1d مع Galcer ، ذوبان biotynlated CD1d - B2وبين عشية وضحاها م المحتضنة في درجة حرارة الغرفة مع فائض three المولي أضعاف من Galcer ، الذائبة في توين -20 بنسبة 0.5 ٪ في برنامج تلفزيوني ، O.9 ٪ كلوريد الصوديوم. Tetramers تتولد عن طريق خلط ل- Galcer تحميل مونومرات CD1d مع فائض 4 أضعاف من المولي PE -- streptavidin مترافق واحتضان لمدة 4 ساعات في درجة حرارة الغرفة. متجر في 48 درجة مئوية.

<ol>
	<li>Benlagha, K., Weiss, A., Beavis, A., Teyton, L., Bendelac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+glycolipipd+antigen-specific+T+cells+using+fluorecent+CD1d+tetramers.">In vivo identification of glycolipipd antigen-specific T cells using fluorecent CD1d tetramers.</a> J. Exp. Med. 191, 1895-1903 (2000).</li><li>Kinjo, Y., Wu, D., Kim, G., Xing, G. W., Poles, M. A., Ho, D. D., Tsuji, M., Kawahara, K., Wong, C. H., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+bacterial+glycosphingolipids+by+natural+killer+T+cells.">Recognition of bacterial glycosphingolipids by natural killer T cells.</a> Nature. 434, 520-525 (2005).</li><li>Naidenko, O. V., Maher, J. K., Ernst, W. A., Sakai, T., Modlin, R. L., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+and+antigen+presentation+of+ceramide-containing+glycolipids+by+soluble+mouse+and+human+CD1d+molecules.">Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules.</a> J. Exp. Med. 190, 1069-1080 (1999).</li><li>Porcelli, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD1+family:+A+third+lineage+of+antigen-+presenting+molecules.">The CD1 family: A third lineage of antigen- presenting molecules.</a> Adv. Immunol. 59, 1-98 (1995).</li><li>Sidobre, S., Kronemberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD1+tetramers:+A+powerful+tool+for+the+analysis+of+glycolipid-reactive+T+cells.">CD1 tetramers: A powerful tool for the analysis of glycolipid-reactive T cells.</a> J. Immunol. 169, 1340-1348 (2002).</li></ol>

في هذا الإجراء ، فإنه يظهر كيفية بدء وينمون وتصيب خلايا الحشرات وكيفية عيار الفيروسة العصوية في استخدام هذه الخلايا. وأهم جوانب هذا الإجراء وصيانة الخلايا ومعرفة عيار الدقيق للالفيروسة العصوية. ذات مرة كنت قد ولدت CD1 tetramers ، يمكن استخدامها كأداة قوية لتحليل الخلايا التائية شحمي...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Express Five SFM</td>
<td> Serum Free Medium</td>
<td>Invitrogen</td>
<td>10486025</td>
<td>&nbsp;</td>
<tr>
<td>High Five TM</td>
<td> Insect Cells</td>
<td>Invitrogen</td>
<td>B855-02</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine Pen strep</td>
<td>Antibiotics</td>
<td>Invitrogen</td>
<td>10378-016</td>
<td>&nbsp;</td>
<tr>
<td>25cm2 TC Flask</td>
<td> Plastic</td>
<td>Fisher</td>
<td>10-126-28</td>
<td>&nbsp;</td>
<tr>
<td>175 cm2 TC Flask</td>
<td> Plug seal flask</td>
<td>Fisher</td>
<td>10-126-8</td>
<td>&nbsp;</td>
<tr>
<td>Erlenmeyer Flask</td>
<td> Cell Culture Flask</td>
<td>Fisher</td>
<td>07 200 672</td>
<td>&nbsp;</td>
<tr>
<td>YM 30 Concenterator</td>
<td>30,000 MWCO</td>
<td>Millipore</td>
<td>UFC 903096</td>
<td>&nbsp;</td>
<tr>
<td>FPLC equipment</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BrA Enzyme and Buffer</td>
<td> Biotinylation Kit</td>
<td>Avidity</td>
<td>BIRA500</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Agarose Column</td>
<td>His Tag Protein Purification Column</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting column</td>
<td> To get Rod of Salt from Protein</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MonoQ Column</td>
<td>Anion Exchange Chromatgraphy</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>S200 Column</td>
<td> Size Exclusion Chromatography</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>alpha-Galcer</td>
<td> Glycolipid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PE- Streptavidin</td>
<td> For conjugating Protein</td>
<td>Molecular Probe</td>
<td> S-866</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a method for production of recombinant mcd1d protein in insect cells.

CD1 البروتينات تشكل الطبقة الثالثة من مستضد تقديم الجزيئات. هم جليكوبروتينات سطح الخلية ، كما عبر عنها سلاسل الثقيلة حوالي 50 كيلو دالتون الغليكوزيلاتي التي ترتبط مع noncovalently المكروغلوبولين beta2 -. انهم ربط الدهون بدلا من الببتيدات. على الرغم من بنيتها يؤكد تشابه البروتينات CD1 MHC إلى الصف الأول والجزيئات الدرجة الثانية تقديم المستضد ، الأخدود mCD1d ضيقة نسبيا ، وعميقة ، ومسعور جدا وفقد اثنين من جيوب ملزم بدلا من جيوب الضحلة الموصوفة لعدة الكلاسيكية MHC - ترميز مستضد تقديم الجزيئات. بناء على تسلسل هذه الأحماض الأمينية ، وهذا الأخدود hydrobphobic يوفر بيئة مثالية لربط مستضدات الدهون. من الطبيعي القاتل T (NKT) استخدام الخلايا TCR على الاعتراف glycolipids ملزمة أو قدمها CD1d. وقد شاركت الخلايا التائية المتفاعلة إلى الدهون التي قدمها CD1 في الحماية ضد الأمراض المعدية والمناعة الذاتية ورفض في الورم. وبالتالي ، فإن القدرة على تحديد وتطهير ، وتتبع استجابة الخلية NKT CD1 التفاعل ذو أهمية كبيرة. جيل tetramers ألفا Galactosyl سيراميد (A - Galcer) مع CD1d وبصيرة هامة في بيولوجيا الخلايا NKT. Tetramers مصنوعة من جزيئات CD1 الأخرى كما تم إنشاؤها وهذه الكواشف الجديدة قد وسعت كثيرا من المعارف وظائف الخلايا التائية الدهون التفاعل ، مع إمكانية استخدامها في رصد استجابة لدهن المستندة إلى اللقاحات وفي تشخيص أمراض المناعة الذاتية وغيرها العلاجات.

Watch this Scientific Journal Video about A Method For Production of Recombinant mCD1d Protein in Insect Cells. at JoVE.com

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

طريقة لتحضير خلايا الحشرات والعدوى لهم الفيروسة العصوية للغرض من إنتاج المؤتلف proteinand mCD1d توليد tetramers mCD1d.

وهناك طريقة للحصول على إنتاج بروتين المؤتلف mCD1d في خلايا الحشرات.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

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10.7K Views.  La Jolla Institute for Allergy and Immunology. طريقة لتحضير خلايا الحشرات والعدوى لهم الفيروسة العصوية للغرض من إنتاج المؤتلف proteinand mCD1d توليد tetramers mCD1d.

Video: A Method For Production of Recombinant mCD1d Protein in Insect Cells.

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : طريقة بسيطة لالتنميط الجيني دقيقة

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

بروتوكول لفج العرض وتقارب اختيار طريق الطعوم البروتين المؤتلف

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

الأمثل والاستفادة من<em&gt; الأجرعية</em&gt; بوساطة إنتاج بروتين عابر في<em&gt; نيكوتيانا</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

كفاءة الإنتاج وتنقية المؤتلف الفئران Kindlin-3 من خلايا الحشرات للدراسات البيوفيزيائية

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

التحقيق البروتين البروتين التفاعلات في الخلايا الحية باستخدام الرنين ضوء بارد نقل الطاقة

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

التعبير البروتين المؤتلف لعلم الأحياء الهيكلي في HEK 293F خلايا نظام التعليق: رواية ويمكن الوصول إليه المنهج

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

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 ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

تحليل مقارن التعبير المؤتلف البروتين في Biofactories مختلفة: البكتيريا، خلايا الحشرات ونظم النبات

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

سم كلوي حقن مكروي في الزرد لنموذج قصور كلوي حاد

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

واجهة المستخدم الرسومية لتتبع بمساعدة برنامج تركيز البروتين في النتوءات الخلوية الديناميكية

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

وهناك طريقة للحصول على إنتاج بروتين المؤتلف mCD1d في خلايا الحشرات.

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