نشكر الدكتور Naidenko للمساعدة في تحسين ظروف refolding. وأيد هذا العمل من قبل المعاهد الوطنية للصحة AI58181 منح البحوث لوران Brossay. سيندي تيا بانه معتمد من قبل المعاهد الوطنية للصحة NRSA AI080230 F31. ستيفاني Terrizzi تيا بانه وسيندي أيضا أن تساهم في هذا العمل.

إعداد الأجهزة إدراج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تطعيم 4 × 500 مل من السل قوارير تحتوي كل منها على 50 ميكروغرام / مل من كربنيسيلين والكلورامفينيكول مع الثقافة بين عشية وضحاها من البكتيريا معربا عن بناء KLRG1 البلازميد. عندما تصل إلى 0.6 Å OD في 600 نانومتر ، وتحدث مع إضافة IPTG M 0.4 و احتضان الثقافة للحصول على 4 ساعات اضافية تهتز عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> Resuspend حصاد الخلايا في درجة الحموضة عازلة إعادة تعليق = 8.0 (50 ملي تريس ، حمض الهيدروكلوريك ، و 25 ٪ (W / V) السكروز ، 1 ملم EDTA ، 10 ملي DTT). ملاحظة : يمكن تجميد الكريات في هذه المرحلة. </li><li style=";text-align:right;direction:rtl"> تجمع ما لا يزيد عن 60 مل من resuspensions الجرثومي في دورق البولي بروبلين. إذا كان ذلك ضروريا لضبط 60 مل مع الشركة المصرية للاتصالات 8.0 درجة الحموضة عازلة ويحرك الخليط بنصف السرعة. </li><li style=";text-align:right;direction:rtl"> إضافة إلى إثارة مزيج قطرة الحكمة : الليزوزيم (النهائي = 1 ملغ / مل) ، MgCl2 (النهائي = 5 ملم) ، 2 ملغ الدناز كنت في الجلسرين 50 ٪ الواردة 75 مم كلوريد الصوديوم ، تريتون - X100 (النهائي = 1 ٪) ، DTT ( النهائي = 10 ملم). </li><li style=";text-align:right;direction:rtl"> يصوتن الحل على الثلج لمدة 1.5 دقيقة ، وأجهزة الطرد المركزي في 5000 دورة في الدقيقة في lysates لمدة 10 دقيقة على 4 درجات مئوية. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> صب وطاف. </li><li style=";text-align:right;direction:rtl"> إضافة 15 مل من الحموضة عازلة غسل = 8.0 (50 ملي تريس ، حمض الهيدروكلوريك ، و 0.5 ٪ تريتون X - 100 ، 100 ملي مول كلوريد الصوديوم ، EDTA 1 ملم ، 1 ملم DTT). </li><li style=";text-align:right;direction:rtl"> على الجليد ، ويصوتن حل ل1.5 دقيقة حتى يتم معلق تماما بيليه. </li><li style=";text-align:right;direction:rtl"> العينات في جهاز الطرد المركزي 5000 دورة في الدقيقة لمدة 10 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> كرر 5 مرات الغسيل. </li></ol></li><li style=";text-align:right;direction:rtl"> 5B مع تكرار غسل العازلة دون الطرد المركزي تريتون ، X - 100 وresuspend في 4 مل من مخزن الشركة المصرية للاتصالات. </li><li style=";text-align:right;direction:rtl"> أخذ الوزن الرطب إدراج الهيئات الطين وتخزينها في المخزن TE بتركيز من 30 إلى 100 ملغ / مل. </li></ol> Refolding من KLRG1 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل 1 لتر من الحموضة refolding العازلة = 8.0 (0.4 - M ارجينين حمض الهيدروكلوريك و 0.1 درجة الحموضة 8-8،3 M تريس ، EDTA 2 ملم ، 0.2 ملم PMSF ، 3 EDTA خالية اقراص مثبطات الأنزيم البروتيني ، 5 و 0.5 ملي GSH GSSG ملم) والبرد إلى 4 درجات مئوية مع التحريك. </li><li style=";text-align:right;direction:rtl"> إعداد الهيئات إدراج للحقن في الخليط للطي : وهي مثالية لجعل 5 10 الحقن مع كل تركيز 0.25 ميكرومتر 0.5 بحيث تركز النهائي من البروتين سوف يكون 2 3 ميكرومتر. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تذوب حجم 50 ملغ من الطين الهيئات إدراجها في GnHCl M 7 مع 10 ملي β - المركابتويثانول. </li><li style=";text-align:right;direction:rtl"> الحفاظ على إدراج الهيئات / خليط GnHCl عند 37 درجة مئوية لمدة 30-40 دقيقة ودوامة كل 5 دقائق لضمان انصهار كامل (الطين سيتضح عندما ذابت تماما). </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي بسرعة قصوى لمدة 30 دقيقة في microcentrifuge عند 4 درجة مئوية ، وطاف لنقل أنبوب الطرد المركزي 15 مل. لا لنقل أي من بيليه ومسود الصغيرة. </li></ol></li><li style=";text-align:right;direction:rtl"> ضبط حجم المخزن المؤقت مع حقن (3 M GnHCl ، 10 NaAcetate ملي ، ودرجة الحموضة 4.2،10 ملي EDTA). </li><li style=";text-align:right;direction:rtl"> حقن 1 مل من الهيئات إدراج المخفف كل ساعة. عندما بالحقن ، ويحرك بسرعة عالية ، إضافة 1 مل من المخفف إدراج الهيئات قطرة قطرة مع 2 ثانية بين كل قطرة. عندما يتم حقن ويحرك بسرعة منخفضة. </li><li style=";text-align:right;direction:rtl"> يستمر التحريك بين عشية وضحاها على سرعة منخفضة في 4 درجات مئوية. </li></ol> تركيز ردود الفعل Refolding <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد بروتوكول ميليبور ق ، والتركيز على رد الفعل L 1 (0.22 ميكرون تصفية) قبل تصفيتها refolding باستخدام خلية وحرك Amicon YM10 الفائق 10K NMWL غشاء (ميليبور) إلى 10 مل ~. </li><li style=";text-align:right;direction:rtl"> التركيز على 2 مل ≤ باستخدام Centriplus YM - 10 10K MWCO (ميليبور). </li></ol> تنقية tetramer KLRG1 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الإعداد AKTAFPLC في 4 درجات مئوية وفقا للأدلة الرعاية الصحية GE. </li><li style=";text-align:right;direction:rtl"> تنقية شكل من مونومر KLRG1 بواسطة اللوني استبعاد حجم باستخدام Sephacryl S - 200 16/60 عالية الدقة العمود (جنرال الكتريك للرعاية الصحية) في HEPES ملي 20 و 150 مم كلوريد الصوديوم. (أنظر الشكل 1). </li><li style=";text-align:right;direction:rtl"> التركيز إلى 1 مل باستخدام Centriplus YM - 10 10K MWCO (ميليبور). </li><li style=";text-align:right;direction:rtl"> استخدام انزيم البيرا وفقا لبروتوكول الطمع s لbiotinylate المونمر KLRG1. </li><li style=";text-align:right;direction:rtl"> يتم التخلص من البيوتين مجانا من حجم الاستبعاد اللوني كما في 2. </li><li style=";text-align:right;direction:rtl"> هو tetramerized KLRG1 جزيء عن طريق خلط KLRG1 مونومر مع 4 أضعاف المولي streptavidin PE الزائدة مترافق دينار بحريني (العلوم البيولوجية). </li></ol> ممثل النتائج <a href="https://www.jove.com/files/ftp_upload/1702/1702fig1.jpg"><img src="/files/ftp_upload/1702/1702fig1tmb.jpg" alt="الشكل 1" border="0" /></a> refolded الشكل 1. نتائج إيجابية من الممثل اللوني FPLC AKTA استبعاد حجم KLRG1. ويبين الرسم البياني انفصال الأحادي (83.52 مل) ، ديمر (56.36 مل) ، وmultimer (36.26 مل) شكل KLRG1 refolded. في ذروة 94.25 مل يمثل تبادل العازلة.

<ol>
	<li>Tessmer, M. S., Fugere, C., Stevenaert, F., Naidenko, O. V., Chong, H. J., Leclercq, G., Brossay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KLRG1+binds+cadherins+and+preferentially+associates+with+SHIP-1.">KLRG1 binds cadherins and preferentially associates with SHIP-1.</a> Int Immunol. 19, 391-400 (2007).</li><li>Grundemann, C., Bauer, M., Schweier, O., von Oppen, N., Lassing, U., Saudan, P., Becker, K. F., Karp, K., Hanke, T., Bachmann, M. F., Pircher, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+identification+of+E-cadherin+as+a+ligand+for+the+murine+killer+cell+lectin-like+receptor+G1.">Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1.</a> J Immunol. 176, 1311-1315 (2006).</li><li>Ito, M., Maruyama, T., Saito, N., Koganei, S., Yamamoto, K., Matsumoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killer+cell+lectin-like+receptor+G1+binds+three+members+of+the+classical+cadherin+family+to+inhibit+NK+cell+cytotoxicity.">Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity.</a> J Exp Med. 203, 289-295 (2006).</li><li>Li, Y., Hofmann, M., Wang, Q., Teng, L., Chlewicki, L. K., Pircher, H., Mariuzza, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+natural+killer+cell+receptor+KLRG1+bound+to+E-cadherin+reveals+basis+for+MHC-independent+missing+self+recognition.">Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.</a> Immunity. 31, 35-46 (2009).</li><li>Nakamura, S., Kuroki, K., Ohki, I., Sasaki, K., Kajikawa, M., Maruyama, T., Ito, M., Kameda, Y., Ikura, M., Yamamoto, K., Matsumoto, N., Maenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K.+Molecular+basis+for+E-cadherin+recognition+by+killer+cell+lectin-like+receptor+G1+(KLRG1).">K. Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1).</a> J Biol Chem. , .</li></ol>

في هذا البروتوكول ، فإنه يظهر كيفية تنقية ، biotinylate وKLRG1 tetramerize. يمكن أن تستخدم لتسمية KLRG1 tetramer KLRG1 يغاندس بواسطة التدفق الخلوي. على الرغم من الظروف refolding سيتعين اختبارها تجريبيا لكل من البروتين ، يمكن tetramerized غيرها من نوع C - lectins باستخدام بروتوكول مماثل. ويمكن استخدام الب?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>BirA Enzyme and Buffer</td>
<td>Biotinylation Kit</td>
<td>Avidity</td>
<td>BIRA500</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitor Tablets, EDTA Free</td>
<td>Reagent</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>or equivalent</td>
</tr>
<tr>
<td>Amicon Stirred Cell</td>
<td>Equipment</td>
<td>Millipore</td>
<td>Model #8400</td>
<td>or equivalent</td>
</tr>
<tr>
<td>YM10 NMWL 10K ultrafiltration membrane</td>
<td>Filtration Supply</td>
<td>Millipore</td>
<td>13642</td>
<td>&nbsp;</td>
<tr>
<td>15 ml YM10 concentrator</td>
<td>Filtration Supply</td>
<td>Millipore</td>
<td>4411</td>
<td>&nbsp;</td>
<tr>
<td>AKTAFPLC</td>
<td>Equipment</td>
<td>GE Healthcare</td>
<td>18-1900-26</td>
<td>&nbsp;</td>
<tr>
<td>Sephacryl S-200 16/60 high-resolution column</td>
<td>Equipment</td>
<td>GE Healthcare</td>
<td>17-1166-01</td>
<td>&nbsp;</td>
<tr>
<td>Strepavidin PE</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>554061</td>
<td>or equivalent</td>
</tr>		</tbody>
		</table>
		</div>

a protocol for the production of klrg1 tetramer

قاتل مستقبلات الخلية G1 lectin مثل (KLRG1) هي عبر الغشاء مستقبلات بروتين سكري من النوع الثاني المثبطة ينتمون إلى الفصيلة C lectin تشبه النوع. KLRG1 موجود بوصفه على حد سواء ونتيجة لمونومر homodimer ثاني كبريتيد مرتبطة. تم العثور على هذه المستقبلات جيدا الحفاظ على خلايا NK الأكثر نضجا وتفعيلها مؤخرا فضلا عن مجموعة فرعية من خلايا تي المستجيب / الذاكرة. باستخدام KLRG1 tetramer فضلا عن أساليب أخرى ، E ، N ، وحددت R - cadherins كما KLRG1 يغاندس. هذه كا + التي تعتمد على خلية خلية التصاق جزيئات تتكون من خارج الخلية التي تحتوي على مجال يكرر كادهيرين الخمسة المسؤولة عن الخلية الخلية والتفاعلات ، ومجال وعبر الغشاء مجال هيولي مرتبط إلى الهيكل الخلوي أكتين 2 وكان جيل من tetramer KLRG1 ضرورية لتحديد يغاندس KLRG1. KLRG1 tetramer هو أيضا أداة فريدة لإلقاء الضوء على الأدوار واللعب كادهيرين KLRG1 في تنظيم الاستجابة المناعية وسلامة الأنسجة.

Watch this Scientific Journal Video about A Protocol for the Production of KLRG1 Tetramer at JoVE.com

A Protocol for the Production of KLRG1 Tetramer

يصف هذا البروتوكول إنتاج tetramer KLRG1 ، الذي هو أداة قوية لتحليل KLRG1 يغاندس.

بروتوكول لإنتاج Tetramer KLRG1

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

a-protocol-for-the-production-of-klrg1-tetramer

Research

JoVE Journal

Biology

9.9K Views.  Brown University. يصف هذا البروتوكول إنتاج tetramer KLRG1 ، الذي هو أداة قوية لتحليل KLRG1 يغاندس.

Video: A Protocol for the Production of KLRG1 Tetramer

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

بروتوكول للتربية البعوض (A. الغامبية)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

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

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

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 ...

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

المستضد CD4 + تصور معين باستخدام خلايا T MHC الدرجة الثانية Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

لإنتاج C. ايليجانس الجينات المحورة عبر Recombineering مع galK إختيار ماركر

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

ومنهاج عمل مريحة والتعبير العام لانتاج البروتينات يفرز من خلايا الإنسان

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

A بروتوكول بسيط لاستخراج اليرقات من Hemocytes البرية

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

بروتوكول SIVQ-LCM لالصك ArcturusXT

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

قياس وتحليل حمض خارج الخلية الإنتاج لتحديد معدل حال السكر

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

بروتوكول إنتاج رواية لتصنيع على نطاق صغير من بروبيوتيك المخمرة للأغذية

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

A متعدد حفرة Cryovial يزيل تجميد قطع أثرية عندما منغمسين الأنسجة العضلية مباشرة في النيتروجين السائل

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...