قسم الدفاع W81XWH - 05 - 1-0005 ، W81XWH - 06 - 1-0495 ، W81XWH - 08-1 0029 وW81XWH - 08-1 - 0356

1. إعداد GST الانصهار بروتين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مصنوعة من مخزونات الجلسرين JM109 البكتيريا المختصة التي تحتوي على أول محطة N - 90 الأحماض الأمينية في المجال rhotekin ملزمة رو (RBD) subcloned في ناقلات pGEX3x BamH1/EcoR1. لا بد منه لضمان كفاءة عالية يتم تنفيذ الخطوات التالية للحصول على كل تجربة. في تجربتنا ، التي أعدت حديثا GST - RBD هو المفتاح لمقايسة GTPase التنشيط قوية ودقيقة. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر (تقريبية ، لا الذوبان) من الأسهم الجلسرين إلى 50 مل أمبير LB وتنمو 37 درجة مئوية خلال الليل مع اهتزاز. </li><li style=";text-align:right;direction:rtl"> تمييع 01:10 LB في 500 مل أمبير وتنمو لمدة 1 ساعة. </li><li style=";text-align:right;direction:rtl"> حمل GST إنتاج البروتين بنسبة 0.1 ملي IPTG لمدة 2 ساعة. </li><li style=";text-align:right;direction:rtl"> توزع بالتساوي في زجاجات الطرد المركزي وأجهزة الطرد المركزي ، Sorval الدوار في 5000 دورة في الدقيقة GSA3 4 درجات مئوية لمدة 20 دقيقة. </li><li style=";text-align:right;direction:rtl"> Resuspend بيليه في زجاجات الطرد المركزي ، و 10 مل من تحلل العازلة *. * الاحتياطي تحلل الجرثومي : السكروز 20 ٪ ، 10 ٪ الجلسرين ، و 50 ملي تريس الرقم الهيدروجيني 8.0 ، 0.2 ملم نا 2 S 2 O 5 ، 2 مم MgCl 2 ، 2 DTT ملم ، إضافة PMSF الطازجة ، بينزاميد ، وأبروتينين leupeptin. </li><li style=";text-align:right;direction:rtl"> يصوتن 2-3 دقيقة (وهذا يعتمد على نوع sonicator. نستخدم سونيك فيشر Dismembranator موديل 500 علامة وضعت في 6 و 50 ٪ دورة). </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي ، Sorvall SS34 ، 20 دقيقة 10000 دورة في الدقيقة في 4 درجات مئوية. عند هذه النقطة يمكن أن يتخذ من قسامة الصغيرة ويديرها SDS - PAGE. يمكن أن يكون coomassie الجل الملون والفرقة واضحة وينبغي على 46 كيلو دالتون. </li><li style=";text-align:right;direction:rtl"> طاف إزالة وإضافة 1 مل من 50 ٪ glutatione - 4B Sepharose الطين. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 30 دقيقة عند 4 درجات مئوية مع التناوب. تغسل 3 مرات مع تحلل العازلة. </li><li style=";text-align:right;direction:rtl"> Resuspend حبات الطين GST-RBD/glutatione-Sepharose إلى 50 ٪ (حوالي 1 مل) مع GST الأسماك العازلة **. ** GST السمك الاحتياطي : الجلسرين 10 ٪ و 50 ملي تريس الرقم الهيدروجيني 7،4 ، و 100 ملي مول كلوريد الصوديوم ، و 1 ٪ NP - 4 ، 2 مم MgCl 2 ، إضافة PMSF الطازجة ، بينزاميد ، وأبروتينين leupeptin </li></ol> 2. GST الانصهار هدم مقايسة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام 50-10 × 10 6 خلية / مقايسة </li><li style=";text-align:right;direction:rtl"> غسل خلايا مرة واحدة في برنامج تلفزيوني الجليد الباردة ، والحفاظ على لوحات على الجليد (نستخدم صحن الزجاج المسطح مملوءة الجليد) وليز مع 0.5 العازلة GST الأسماك مل. </li><li style=";text-align:right;direction:rtl"> احتضان 5 دقائق على الجليد ، وخلايا الحصاد باستخدام المزيل شرطي المطاط أو شقة ، ونقل إلى microfuge الأنابيب وأجهزة الطرد المركزي 20000 دورة في الدقيقة 5 دقائق على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> طاف لنقل أنبوب microfuge الطازجة. تأخذ 50 ميكرولتر لتحديد رو الكلي (أي الناتج المحلي الإجمالي GTP + ملزمة). </li><li style=";text-align:right;direction:rtl"> إضافة 0.5 مل GST-RBD/glutatione-Sepharose الخرز واحتضان عند 4 درجة مئوية ، بين عشية وضحاها مع التناوب. </li><li style=";text-align:right;direction:rtl"> يغسل مع 6X GST الأسماك العازلة. </li><li style=";text-align:right;direction:rtl"> Resuspend الخرز في 40 ميكرولتر العازلة عينة - 2X Laemelli عند هذه النقطة يجب أن يتم تحديد تركيز البروتين الكلي قسامة رو المتخذة في 2.4 و 30 ميكروغرام أعدت ليتم تشغيلها على هلام مع العينة التنشيط المقابلة. </li><li style=";text-align:right;direction:rtl"> عينات يغلي عند 90 درجة مئوية لمدة 5 دقائق ، طرد المركزي لفترة وجيزة ، واتخاذ طاف (أي تجنب الخرز) تحميل وتشغيل المعامل على 12 ٪ جيد 40-20 SDS - PAGE هلام. </li><li style=";text-align:right;direction:rtl"> تشغيل هلام في أي أعلى من 150 فولت ، ونقل إلى استخدام غشاء Towbin العازلة الحالية والمستمرة من 100 دبليو. </li><li style=";text-align:right;direction:rtl"> كتلة الغشاء بمحلول الحليب / TBST 5 ٪ ، والتحقيق مع RhoC - specfic الأضداد. </li></ol> 3. ممثل النتائج وينبغي أن تنتج نتائج جيدة في شريط واحد حوالي 22 كيلو دالتون. النتائج السيئة تنتج نطاقات متعددة أو خلفية عالية. هذا يدل على تدهور البروتين ، وغسل العينات غير مكتملة أو استخدام القديمة GST - RBD. <img src="/files/ftp_upload/2083/2083fig1.jpg" alt="الشكل 1" /> الشكل 1. تظهر ثلاثة خطوط منفصلة خلية التعبير عن مستويات مختلفة من GTPase RhoC لإظهار مستويات منخفضة من RhoC نشطة وإجمالا ، مستويات عالية من RhoC النشط والكلي أو RhoC لا. جردت ثم وصمة عار reprobed مع أجسام مضادة لهذا الجين أكتين منزل حفظ لتكون بمثابة سيطرة التحميل للبروتين RhoC الإجمالي. <img src="/files/ftp_upload/2083/2083fig2.jpg" alt="الشكل 2" /> الشكل 2. نطاقات متعددة أو خلفية نتائج عالية من البروتين التدهور ، وغسل العينات غير مكتملة أو استخدام القديمة GST - RBD.

<ol>
	<li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. W., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase,+a+novel+transforming+oncogene+for+human+mammary+epithelial+cells+that+partially+recapitulates+the+inflammatory+breast+cancer+phenotype.">RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype.</a> Cancer Res. 60 (20), 5832-5838 (2000).</li><li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+overexpression+modulates+induction+of+angiogenic+factors+in+breast+cells.">RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells.</a> Neoplasia. 2 (5), 418-425 (2000).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+RhoC+GTPase-induced+inflammatory+breast+cancer+phenotype+by+treatment+with+a+farnesyl+transferase+inhibitor.">Reversion of RhoC GTPase-induced inflammatory breast cancer phenotype by treatment with a farnesyl transferase inhibitor.</a> Mol Cancer Ther. 1 (8), 575-583 (2002).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+activated+protein+kinase+pathway+is+involved+in+RhoC+GTPase+induced+motility,+invasion+and+angiogenesis+in+inflammatory+breast+cancer.">Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer.</a> Clin Exp Metastasis. 19 (4), 301-311 (2002).</li><li>Yao, H., Dashner, E. J., van Golen, C. M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+is+required+for+PC-3+prostate+cancer+cell+invasion+but+not+motility.">RhoC GTPase is required for PC-3 prostate cancer cell invasion but not motility.</a> Oncogene. 25 (16), 2285-2296 (2006).</li><li>Hall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+Collagen+receptor+(alpha2beta1)+signaling+promotes+prostate+cancer+cell+invasion+through+RhoC+GTPase.">Type I Collagen receptor (alpha2beta1) signaling promotes prostate cancer cell invasion through RhoC GTPase.</a> Neoplasia. 10 (8), 797-803 (2008).</li><li>Sequeira, L., Dubyk, C. W., Riesenberger, T. A., Cooper, C. R., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+PC-3+prostate+cancer+cell+morphology,+invasion+and+tumor+cell+diapadesis.">Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapadesis.</a> Clin Exp Metastasis. 25, 569-579 (2008).</li><li>van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL2+Induces+Prostate+Cancer+Transendothelial+Cell+Migration+via+Activation+of+the+Small+GTPase+Rac.">CCL2 Induces Prostate Cancer Transendothelial Cell Migration via Activation of the Small GTPase Rac.</a> J Cell Biochem. 104, 1587-1597 (2008).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Int J Cancer. , (2010).</li><li>Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A., Collard, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+downregulates+Rho+activity:+reciprocal+balance+between+both+GTPases+determines+cellular+morphology+and+migratory+behavior.">Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.</a> J Cell Biol. 147 (5), 1009-1022 (1999).</li></ol>

الإعلان عن أي تضارب في المصالح.

الخطوة الأكثر أهمية من هذا الإجراء هو الجيل الجديد من GST - RBD. عدم إيلاء اهتمام وثيق لهذه الخطوة هو السبب الرئيسي للفشل. جميع الخطوات ، من جيل لGST - RBD (1.2) إلى حضانة بين عشية وضحاها مع lysate الخلية (2.5) ، ينبغي أن يتم في يوم واحد. خطوة أخرى حاسمة هي التأكد من ان هناك ما يكفي من ا?...

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<td>Gluthione-sepharose 4B beads</td>
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<td>Criterion 4-20% Tris-HCl gels</td>
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rhoc gtpase activation assay

RhoC GTPase وقد تماثل 91 ٪ إلى GTPase RhoA. بسبب انتشاره في الخلايا ، وقد وضعت العديد من الكواشف والتقنيات اللازمة لGTPase RhoA. ومع ذلك ، أعرب RhoC GTPase في خلايا السرطان النقيلي عند مستويات منخفضة نسبيا. لذا ، وضعت بعض الكواشف RhoC محددة. وقد تكيفت لدينا فحص للكشف عن التنشيط رو GTPase RhoC. هذا الأسلوب يستخدم لضريبة السلع والخدمات ، رو الانصهار المجال ملزمة بالانسحاب من البروتين النشط RhoC GTPase. بالإضافة إلى ذلك ، يمكننا أن محصول البروتين الكلي في بداية الفحص لتحديد مستويات من المجموع (GTP الناتج المحلي الإجمالي وملزمة) RhoC GTPase. وهذا يسمح لتحديد GTPase RhoC النشطة مقابل الإجمالي في الخلية. وقد وضعت العديد من الإصدارات التجارية من هذا الإجراء ومع ذلك ، هي الأمثل مجموعات تجارية لGTPase RhoA وعادة لا تعمل بشكل جيد لGTPase RhoC. تم تعديل أجزاء من الفحص وكذلك وضع الضد RhoC محددة.

Watch this Scientific Journal Video about RhoC GTPase Activation Assay at JoVE.com

RhoC GTPase Activation Assay

هذا البروتوكول يستخدم لهدم مقايسة لتحديد مستويات GTPase RhoC نشطة داخل الخلايا.

RhoC الفحص GTPase التنشيط

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Research

JoVE Journal

Biology

12.3K Views.  University of Delaware. هذا البروتوكول يستخدم لهدم مقايسة لتحديد مستويات GTPase RhoC نشطة داخل الخلايا.

Video: RhoC GTPase Activation Assay

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

مقابلة : شحمي سكري تقديم المستضد بواسطة CD1d والعلاجية المحتملة لتنشيط الخلايا NKT

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

تسجيلات لتنشيط الدائرة العصبية في التصرف بحرية حيوانات

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

الأبهر خاتم الفحص

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

والعزلة في المختبر تفعيل انواع معينة ايليجانس الحيوانات المنوية

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

تفعيل موت الخلايا المبرمج من قبل Microinjection هيولي من السيتوكروم ج</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

الخلايا Refolding الفحص

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

أميد الهيدروجينية / الديتريوم الطيف الكتلي تبادل و- TOF MALDI تحليل Pak2 التنشيط

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

واحدة جزيء التصوير من تنظيم الجينات<em&gt; في الجسم الحي</em&gt; استخدام التنشيط بواسطة Cotranslational انشقاق (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. ايليجانس الانجذاب الكيميائي الفحص

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

مقارنة الانجذاب من البروتينات ملزم GTPase باستخدام فحوصات المنافسة

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...