This work was supported by NIH grants ARRA RO3 AI078145 and 1SC3GM113743 to RZ.

1. Hücre Özüt Hazırlama <ol><li> 20 mM HEPES pH7.4, 100 U / ml penisilin / ml streptomisin, 5 ug / ml heme 100 ug ile takviye edilmiş M199 1 x yapılmış bir ortam içinde 26 ° C &#39;de hava geçirmez kapak ile sızdırmaz steril plastik şişe Leishmania hücreleri büyümek çalkalamadan, 0.35 g / L NACO 2H, 0.1 mM adenin, ve 2 ug / ml biopterin. Bunlar 1-2 x 10 7 / ml&#39;lik bir hücre yoğunluğuna ulaştığında 4 ° C &#39;de 5 dakika süre ile 1500 g&#39;de santrifüje edilerek hücreler hasat. </li><li> Bir pipet ile süpernatant atılır ve soğuk fosfat tamponlu serum fizyolojik kültürün hacmi (137 mM NaCI, 2.7 mM KCI yarısı bir serolojik pipet hücre topağın tekrar askıda bırakılmasıyla yıkama hücreleri, 10 mM Na-2 HPO 4, 1.8 mM KH 2 PO 4, pH 7.4). BL2 güvenlik kurallarına göre hücre süpernatantlar bertaraf edin. </li><li> Hücrelerin tekrar santrifüj 4 ° C&#39;de 5 dakika boyunca 1500 g&#39;de. Bir pipet ile süpernatant atın. T Devamya da bir sonraki adıma o -80 ºC (üç ay) uzun süreli saklama için sıvı nitrojen içinde hücre pelletini dondurmak oturtun. </li><li> 2x lizis tamponu (0.5 M sukroz, 0.1 M TrisHCl, pH 7.5, 2 mM EDTA ve 2 x proteaz inhibitör kokteyli) hazırlayın ve buz üzerinde 4 ° C&#39;de tutun. </li><li> 2x lizis tamponu eşit hacimde (taze veya dondurulmuş) hücre pelletini. Cam boncuklar 1x hacmi ekleyin. Vorteks kuvvetli bir şekilde 10 dakika boyunca 4 ° C&#39;de. </li><li> 1x liziz tamponu 2 hacim ekleyin ve karıştırın. 10 dakika boyunca 4 ° C&#39;de 1500 x g&#39;de santrifüjleyin hücre ekstreleri kesintisiz hücre ve çekirdeklerin: pelet. </li><li> Bir taze soğuk bir santrifüj tüpü içine pipetle aktarın ve süpernatant deney tamamlanana kadar buz üzerinde hücre ekstreleri tutun. </li></ol> 2. Bu tür Bikinkoninik Asit Testi Protein Tahmin Kiti Kullanımı Cep Özü Protein Konsantrasyon belirleyin <ol><li> Bisinkoninik asit hazırlanması (BCA) çözeltisi BCA ve bakır (II) karıştırılması ile (1 mL / tüp) s49 oranında ulfate: 1 (v / v). </li><li> Protein standartlarını hazırlayın 0, 10, 20, 30, 40, 50 ve BCA solüsyonun 1 ml&#39;lik alikolar içinde bir 10 mg / ml bovin serum albümini (BSA) stok solüsyonu seyreltilerek 60 ug / ml olmuştur. </li><li> Tekrarlardaki BCA çözeltisi 1 ml hücre ekstrelerinin 2 ul ekleyin. Önceden ısıtılmış 60 ° C su banyosu içinde 10 dakika için standartlar ve protein örnekleri inkübe edin. </li><li> Aktarım numuneler 3 dakika boyunca buz. 562 nm bir dalga boyunda bir spektrofotometre ile standart ve protein örnekleri absorbansı ölçülür. </li><li> Üreticinin protokolünde tarif edildiği gibi, bir referans olarak BSA standardı kullanılarak hücre ekstrelerinin protein konsantrasyonu hesaplanır. 1x lizis tamponu ile mi, 10 mg / bir protein konsantrasyonuna hücre ekstreleri seyreltilir. </li></ol> Tüp başına 200ul 3. Enzimatik Deneyi NOT: Bir kimyasal kaputu aşağıdaki adımları uygulayın. <ol><li> 15 ml nüsha halinde her bir numune test edinkonik tüp. Tüp başına 20 ul 1 M TrisHCl pH 7.5 hazırlayın ve buz üzerinde tutmak. Her bir tüp, oda sıcaklığında, (çözelti durdurma 1 (h / h) 1) kloroform / metanol 2 ml hazırlayın. </li><li> Pipet, buz üzerinde, her 15 ml konik bir tüp içinde 1 M TrisHCl pH 7.5, 20 ul. </li><li> Burada radyasyon güvenlik kurallarına uyun. SAM 50,06 uM toplam tüp başına [metil- 3 H] adenosil-L-metionin ve 50 uM soğuk SAM - 0.06 uM (0.2 uCi), S eşdeğer ekleyin. Soğuk su ul x ekle x = 200- (20 (+ hücre ekstreleri için 20 () + soğuk ve radyoaktif SAM hacmi) tamponu için) tüp başına. </li><li> Önceden ısıtılmış 30 ºC su banyosunda her konik tüp aktarın. Reaksiyonu başlatmak için her bir tüpe, hücre ekstreleri (proteinin 200 ug eşdeğer) 20 ul ekle. İstediğiniz zaman (0-45 dakika) inkübe edin. </li><li> Kloroform / metanol 2 ml ilave ederek reaksiyonu durdurun (1: 1; v / v; durdurma çözeltisi) her tüpe. (Oda sıcaklığına 20-25 konik tüp transfer6 ° C). </li></ol> 4. Lipid Ekstraksiyon NOT: Bir kimyasal kaputu aşağıdaki adımları uygulayın. <ol><li> Enzimatik reaksiyon örneğini içeren her tüpe su 700 ul ekle. Kuvvetlice 30 saniye boyunca girdap. Oda sıcaklığında 5 dakika boyunca 1500 x g&#39;de santrifüjleyin su fazının organik ayırmak için. </li><li> Bir pipet yardımıyla yeni bir 15 ml konik tüp içine alt organik fazı aktarın. 30 saniye boyunca kuvvetli bir şekilde boru ve vorteks içeren her &quot;düşük değerli faz&quot; su 1 ml ilave edilir. Tekrar santrifüj 5 dakika boyunca 1500 x g&#39;de su fazının organik ayırmak için. </li><li> Bir pipet, bir sintilasyon tüpüne alt organik faz transfer edin. N2 akışı altında kuru örnekleri. Olmayan anonim radyoaktif SAM ve radyasyon kurallarına göre radyoaktif konik tüpler içeren su fazları bertaraf edin. </li><li> 2 ml / sintilasyon sıvısı tüp ekleyin. Bir sintilasyon cou ile radyoaktiviteyi ölçünnter üreticilerin protokol ve enstrümanın kullanıma göre. </li><li> Aşağıdaki genel denklemi kullanarak nmol / mg protein enzimatik aktivite hesaplanır cpm x değeri 10 3 x [SAM (mM) &#39;nin toplam (radyoaktif ve soğuk) konsantrasyonu], radyoaktif SAM (Ci / mmol) x x 5 spesifik aktivitesi [radyoaktif SAM konsantrasyonu (mM)] Not: Yukarıdaki protokole bağlı SAM konsantrasyonu ya da bir protein bağlı PEMT tahlili için değiştirilebilir. SAM, konsantrasyona bağlı enzimatik deney için, zaman ise, protein bağlı PEMT tahlilinde, SAM konsantrasyonu için tahlil eklenir sabit (lineer aralığında 15 dakika) ve soğuk SAM çeşitli miktarlarda tutulur (biz mM 0.05 seçtik) ve zaman sabiti (15 dakika) tutulur. Ayrıca tampon çözeltisinin pH değeri, bazı PEMT enzimler farklı optimal pH değerine sahip olması gerektiği gibi değiştirilebilir. </li></ol>

<ol>
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Herhangi bir çıkar çatışması ilan

Bu basit, pratik PEMT tahlil, bir protein kaynağı olarak bütün hücre ekstresi kullanarak PE üzerine SAM radyoaktif metil gruplarının transferi sonucu PE metillenmiş formlarının ölçümü sağlar. Bu, hızlı, hassas ve tekrarlanabilir ve saflaştırılmış enzimler 17 için de uygundur. Ilgi metiltransferaz yerine PE 12,13,18,19 daha Bu yüzeylerde özgü olup olmadığını monometil veya dimetil-PE tahlilinde eklenebilir. Saflaştınldı PEMT enzim kullanılırsa, PE tahlilinde eklen...

Fosfatidiletanolamin metiltransferaz (PEMT) PEMT enzimler monometil-PE, dimetil-PE ve / veya vermek için PE, monometil-PE veya dimetil-PE üzerine metil grubu donörü S -adenosylmethionine (SAM) ile bir veya daha fazla metil grubu kovalent bağlanmasını katalize eden bir fosfatidilkolin (PC). Bu enzimler, hayvan hücreleri ve mantarlar hemen hemen her zaman görülmektedir. Ayrıca, bazı bitkilerin 1 ve bakterilerin yaklaşık% 10, ökaryotlarda 2 ile etkileşim özellikle bulunabilir. PEMTs değil, aynı zamanda diğer önemli hücresel fonksiyonları yerine getirerek hayvan hücrelerinde ana lipid sınıf PC, üretimine katkıda bulunarak hücre biyolojisine alakalı. Memelilerde, PEMTs esas olarak çok düşük yoğunluklu lipoprotein normalin üzerinde bir salgılama için gerekli olan karaciğer olarak ifade edilmiştir ve ayrıca diyet indükte obeziteye 3, ateroskleroz 4 katkıda bulunur ve insülin karşıance 5. Buna ek olarak, bir memeli PEMT da her ne kadar düşük seviyelere, adipositler ile ifade edilmiştir ve yağ depolanması 6, 7 katılırlar. Kanser gelişimi 8, 9 apoptoz ve hücre büyümesi 10&#39;da PEMT rolü de ortaya konmuştur. Bakterilerde PEMT enzimler ana bitki 11, normal hücre çoğalması 2, virülans 2 ve simbiyoz için önemli olduğu gösterilmiştir. Mevcut protokolün amacı ve gerekçesi enzimi arındırmak gerek kalmadan bütün hücre ekstrelerinden PEMT aktivitesini ölçmektir. İki farklı protokoller PEMT aktivitesini ölçmek için, geliştirilmiştir. İlk ve en yaygın olanı, bu makalenin konusu olan PE, üzerine radyoaktif SAM trityumlu metil grubunun transferini ölçer. Bu protokol, ilk olarak bir underst elde etmek için maya 12 ve memeli hücrelerinde (karaciğer) 13 den PEMT aktivitesini ölçmek için, geliştirilmiştirBu hücrelerde PC biyosentezinin anding yanı sıra, bu enzim özgüllüğünü belirlemek için. Daha sonra, bu teknik ve tek hücreli parazitlerin 14 (15 olsa deneyi için bir bazik pH değeri kullanılarak) bu tür bakteri 2 gibi diğer hücre tiplerinde uygulanmıştır. Bu teknik, bütün hücre hülasaları hem de saflaştırılmış enzim ile birlikte kullanılabilir ve potansiyel olarak herhangi bir hücre ekstresi sistemine uygulanabilir. Olmayan bir radyoaktif deneyi de S -adenosylhomocysteine, SAM 16 metillenmenin ürünün enzimatik miktarının dayandığı dizayn edilmiştir. Bu radyoaktivite içermeyen olarak ikinci deney daha uygun olabilir ancak saflaştınldı enzimler için uygundur.

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	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">S-[Methyl-3H]adenosyl-L-methionine (specific activity of 5-15 Ci/mMole)</td>
			<td style="width:107px;">Perkin Elmer</td>
			<td style="width:119px;">NET155050UC</td>
			<td style="width:188px;">Aliquot the reagent and freeze at -20 &#176;C; follow radiation safety guidelines while using this reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Roche Life Sciences</td>
			<td style="width:119px;">11836170001</td>
			<td>dilute it fresh&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed, 425-600 mm</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bicinchoninic acid solution</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">B9643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Copper (II) sulfate</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">C2284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scintillation counter MicroBeta2 with 1-detector</td>
			<td style="width:107px;">Perkin Elmer&#160;</td>
			<td style="width:119px;">2450-0010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectrophotometer Biomate 3</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td>840208300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA stock solution (10 mg/ml)</td>
			<td style="width:107px;">New England Biolabs</td>
			<td style="width:119px;">B9001S</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Scintillation liquid&#160;</td>
			<td style="width:107px;">Research Product International Corp</td>
			<td style="width:119px;">111198</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">S-(5&#39;-Adenosyl)-L-methionine chloride (hydrochloride)&#160;</td>
			<td style="width:107px;">Cayman Chemicals</td>
			<td>13956</td>
			<td style="width:188px;">dilute the reagent in 20 mM HCl and freeze aliquots at -80 &#176;C&#160;</td>
		</tr>
	</tbody>
</table>

in vitro assay to measure phosphatidylethanolamine methyltransferase activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

Şekil 1, bir alt-tabaka olarak endojen PE kullanılarak, bir enzim kaynağı olarak Leishmania bütün hücre ekstresi ile gerçekleştirildiği zamana bağlı PEMT deneyi göstermektedir. Organik faz içinde radyoaktivite miktarı, sintilasyon sayımı ile nicelendirildi. Elde edilen PE numaraları üzerine transfer tritiye metil gruplarının miktarını hesaplamak için kullanıldı. PEMT aktivitesi yaklaşık 20 dakika boyunca doğrusal olmuştur. Daha sonr...

Watch this Scientific Journal Video about In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity at JoVE.com

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Vitro Tahlil Fosfatidiletanolamin Metiltransferaz Aktivite Tedbir

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

in-vitro-assay-to-measure-phosphatidylethanolamine-methyltransferase

Research

JoVE Journal

Biology

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

9.8K Görüntüleme.  St John's University. The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine. 

Video: In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

Işık Parlaklıkları değişiklikler Zebra balığı, Duyarlılık Tedbir Davranış Testi

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biyoloji

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Bir In vitro FluoroBlok Tümör Invasion Testi

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Protein Membran Kaplama Testi: Çözünür ve çözünmez Proteinler Arasında Etkileşim Test Protokolü In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Tümör Hücrelerinin vaskülojenik Faaliyet değerlendirin bir Matrigel Tabanlı Tüp Oluşumu Testi

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2 Kinaz Aktivitesi Assaying In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro Metilasyona Tahlil Protein Arginin metilasyon Okuyacak

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Kantitatif İmmünofloresan Deneyi sentrozomlar Protein Düzeyleri Varyasyon Tedbir

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Sistemik Oksidatif Stresin bir Dolaylı Tedbir olarak Comet Assay

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

Ramsay Testi Kullanımı sıvı salgılanması ve İyon Flux fiyatlar Tedbir<em&gt; Drosophila melanogaster</em&gt; Malpighi Tübül

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...