Hazırlık Talimatı Kuluçka Tampon, Assay Tampon ve 0.6 N TCA Çözüm kullanıma hazır çözümler olarak sağlanmaktadır. FITC Kazein Yüzey tekrarlanan donma-çözülme döngüleri önlemek için vardıklarında küçük birimlere kısım FITC Kazein Yüzey tavsiye edilir. FITC Kazein Yüzey tekrarlanan donma-çözülme döngüsüne maruz kalırsa, arka planda hafif bir artış hassasiyet düşürücü, böylece meydana gelecektir. Alikotları, -20 ° C&#39;de saklanan ve ışıktan korunmalıdır. Her bir örnek, boş, ya da kontrol reaksiyon FITC Kazein Yüzey 20 ul gerektirir. Freeze / Çözülme yanı sıra kuvvetli karıştırma veya sallayarak FITC, okuma ve daha az substrat proteaz bölünme için kullanılabilir hale gelmesine neden yüksek bir arka plan ile sonuçlanan Kazein ayrı neden olabilir. Floresein izotiyosiyanat (FITC) Kontrol Çözüm Testi Tampon FITC kontrolü, uygun konsantrasyonda sulandırılmış olabilir. Bu çözüm, taze ve ışıktan korunmalıdır. Final tripsin Kontrol Çözümü - 1 mM HCl 100 ul tripsin, Proteaz Kontrolü (Ürün Kodu T 6567) flakon. Tripsin çözülmüş olduğundan emin olmak için kısa bir süre karıştırın. Kuluçka Tampon 900 ul ekleyin ve iyice karıştırın. Alternatif olarak, eğer tahlil için istenen diğer tamponlar kullanılabilir. Tripsin son çalışma konsantrasyonu 20 mcg / ml. Tripsin Kontrol Çözüm hazırlamak için hazır olduğunuzda, bir kısım Kuluçka Buffer (9 parça tampon 1 yarı asitlendirilmiş tripsin) doğru miktarda asidik tripsin çözüm birleştirir. Tripsin asidik koşullar altında saklanması tripsin istikrarı artırır. Tripsin kontrol çözümü ısıya duyarlı ve düşük konsantrasyonlarda sabit değildir. Hazırlanan bu çözümler depolama ve istikrarı için kit teknik bülten gözden geçirin. Prosedür <ol><li> Bir kerede tüm kontrolleri, boşlukları ve numuneleri hazırlamak için en kolayıdır. Her bir test örneği için, bir mikrosantrifüj tüp 20 ul Kuluçka Buffer, 20 ul FITC Kazein Yüzey ve test örneği 10 ul ekleyin. Yüksek proteaz aktivitesi test örnekleri, örnek seyreltme gerekebilir. </li><li> Bir mikrosantrifüj tüp 20 Kuluçka Tampon, 20 ul FITC Kazein Yüzey ve kontrol örneği 10 ul ul ekleyerek uygun kontrol örneklerinde (Kontrol Örnekler) hazırlayın. </li><li> Bir mikrosantrifüj tüp 20 Kuluçka Tampon, 20 ul FITC Kazein Yüzey ve ultra saf su 10 ul ul ekleyerek boş bir örnek hazırlayın. </li><li> Her bir tüp ve inkübe 37 ° C&#39;de 60 dakika süreyle karanlıkta yavaşça karıştırınız. Türbülansı yüksek floresan arka plan neden olabilir, çok güçlü bir şekilde karıştırın ve testin duyarlılığını azaltmak için dikkatli olun. Not: İnkübasyon süresi duyarlılığını artırmak için 24 saate kadar uzatılabilir. Yüksek floresan arka önde gelen, 24 saat FITC-Kazein düşmeye başlayabilir aşmayacak şekilde dikkatli olun. </li><li> Inkübasyondan sonra her mikrosantrifüj tüpe 0,6 N Trikloroasetik Asit Çözeltisi 150 ul ekleyin. TCA çok aşındırıcı ve uygun koruyucu ekipman giyerek ele alınmalıdır. </li><li> 37 ° hafifçe karıştırın ve inkübe ° C de 30 dakika süreyle karanlıkta. </li><li> 10.000 x g. az 10 dakika süreyle tüpleri Santrifüj Süpernatant asit çözünür, FITC etiketli parçaları içeren ve floresan ölçümü için kullanılır. </li></ol> Floresan Ölçümler Bu yöntemler ölçekli veya aşağı enstrümantasyon mevcut şartlara göre olabilir. Uygun kontrol örnekleri ile hazırlanan standart eğri karşılaştırma için, her bir test örneği değeri (FLUtest) boş bir örneği (FLUblank) floresan okuma çıkarın. Hendekler <ol><li> Pipet 10 süpernatant (adım 7) ve uygun bir küvet içine Testi Tampon 1 ml ul ve hafifçe karıştırın. Not: süpernatant ve Analiz Tampon çözüm floresan ölçüm önce 24 saate kadar 2-8 ° C&#39;de karanlıkta saklanabilir. </li><li> 485 nm eksitasyon ile floresan kaydetme ve izleme emisyon dalga boyu 535 nm. </li></ol> Multiwell Tabaklar <ol><li> Pipet 10 ul supernatant (adım 7) ve uygun bir tüp veya şişenin içine Testi Tampon 1 ml ve hafifçe karıştırın Not: süpernatant ve Analiz Tampon çözüm, 2-8 ° C&#39;de karanlıkta saklanabilir floresan ölçüm 24 saat önce için. </li><li> Siyah 96 plaka iyi 200 ul aktarın. 485 nm eksitasyon ile floresan kaydetme ve izleme emisyon dalga boyu 535 nm. Veya </li></ol><ol><li> Pipet süpernatantı 2 ul (adım 7) ve Test B 200 ulsiyah 96 plaka bir kuyuya uffer. Not: süpernatant ve Analiz Tampon çözüm floresan ölçüm önce 24 saate kadar 2-8 ° C&#39;de karanlıkta saklanabilir. </li><li> 485 nm eksitasyon ile floresan kaydetme ve izleme emisyon dalga boyu 535 nm. </li></ol> Kontrol Örnekleri Tripsin Kontrol Çözüm tahlil saptama sınırı belirlemek, ya da genel bir standart eğri oluşturmak için, düzgün gerçekleştirirken onaylamak için kullanılabilir. Farklı, özel bir proteaz testi için, uygun inkübasyon tamponunda proteaz içeren bir kontrol solüsyonu hazırlamak için tavsiye edilir. Testin algılama limiti, boş bir örneği ile elde edilen değerin üzerinde önemli bir floresan okuma üretir proteaz miktarı. Algılama sınırı enstrümantasyon hassasiyetine bağlı olarak değişir. Tripsin Kontrol Çözüm Seri dilüsyonları kontrol çözümleri oluşturmak için kullanılan olabilir. Boş bir örneği ile elde edilen değerin% 120 okuma eşit olarak anlamlı kabul edildi. Rutin, tripsin 5 ng algılama sınırı Bu işlem ile elde edildi. Her deney en az bir 5 ng tripsin kontrolü ile çalıştırmak olması tavsiye edilir. 0.5 mg / ml &#39;lik bir konsantrasyon ile tripsin kontrol çözümü tayininde tripsin istenen 5 ng neden olacaktır. Tripsin Kontrol Çözümü (20 mg / ml) 0.5 mg / ml kontrol çözümü, yani bir parçası Kuluçka Tampon 39 parça tripsin Kontrol Çözüm sonuçları 40 kat seyreltme. <img src="/files/ftp_upload/1514/figure1.jpg" alt="Şekil 1" /> Şekil 1 (tripsin Faaliyet Standart Eğrisi): tripsin Kontrol Çözümü (20 mg / ml) (bkz. Şekil 1) seri dilüsyonları yaparak standart bir eğri oluşturmak için kullanılır. Her bir kontrol örneği floresan okuma boş örnek floresan okuma (FLUblank) her kontrol örneği (FLUcontrol) değeri çıkarılarak giderilmiştir. 0.15 mcg / ml (1.5 ng) 2.5 mg / ml (25 ng) arasında değişen bu seti ve kontrol örnekleri kullanarak tripsin (Ürün Kodu T 6567) için tipik bir standart eğri. Bu eğri açıklanan prosedürü izleyerek, adım 4 için 30 dakikalık bir inkübasyon süresi oluşturulmuştur. Multiwell plaka Floresan ölçümleri yapıldı. Floresein izotiyosiyanat mümkün alet kalibrasyon kontrolü (uygun üreticinin talimatlarına bakınız) veya FITC sinyal doğrusallık aralığı belirlenmesi sağlanır.

<ol>
	<li>Twining, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+Isothiocyanate-Labeled+Casein+Assay+for+Proteolytic+Enzymes.">Fluorescein Isothiocyanate-Labeled Casein Assay for Proteolytic Enzymes.</a> Anal. Biochem. 143, 30-34 (1984).</li></ol>

Yazar, bu yazıda kullanılan reaktifler ve araçları üreten Sigma Aldrich bir çalışanı.

Biz sadece biyolojik bir örnek proteaz aktivitesi tespit etmek için Proteaz Floresan Algılama Kit kullanarak bir örnek tahlil göstermiştir. Bu kit, çok çeşitli fizyolojik uygulamalarda bulundu proteazlar tespit etmek için optimize edilmiştir. Serin, sistein, metalli ve aspartik proteazlar tespiti için uygundur, ancak yapılan değişiklikler bazı spesifik proteazlar algılamak için gerekli olabilir.

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments (optional)</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PF0100|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12346">Protease Fluorescent Detection Kit</a></td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>PF0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H1758|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12347">Hydrochloric Acid</a></td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>H1758</td>
<td>&nbsp;</td>
</table>

use of the protease fluorescent detection kit to determine protease activity

Proteaz Floresan Algılama Seti sağlamaktadır hazır proteaz aktivitesi varlığını tespit için kullanımı reaktifler. Bu basit bir tahlil proteaz aktivitesi tespit etmek için kazeinin, substrat olarak floresein izotiyosiyanat (FITC) ile etiketlenmiş kullanır. Proteaz aktivitesi asidik koşullar altında çökelek değil küçük parçaları, içine FITC etiketli kazein substrat bölünme ile sonuçlanır. Proteaz örnek ve yüzey inkübasyondan sonra, reaksiyon trikloroasetik asit (TCA) eklenmesi ile asitlendirilmiş. Bu karışım daha sonra bir pelet ve çözelti içinde kalan küçük, asit çözünür parçaları oluşturan sindirilmemiş substratı ile santrifüj edilir. Süpernatant nötralize edilir ve FITC etiketli parçaları floresan ölçülür. Açıklanan kiti prosedür yaklaşık 0.5 mg / ml (tripsin 5 ng tahlil eklenen) bir konsantrasyon tripsin proteaz kontrol algılar. Bu duyarlılık, daha uzun bir kuluçka süresi, 24 saate kadar artırılabilir. Mikrosantrifüj tüpler tahlil yapılır ve prosedürleri Küvet veya multiwell plakaları kullanarak floresan tespiti için verilmektedir.

Watch this Scientific Journal Video about Use of the Protease Fluorescent Detection Kit to Determine Protease Activity at JoVE.com

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

Proteaz Floresan Algılama Kit Florimetri kullanarak proteaz aktivitesi ölçümü için tasarlanmıştır. Ayrıca eser miktarda proteaz kontaminasyon tespiti için uygundur. Yöntem FITC etiketli kazein substrat özel bir formülasyon proteolitik hydroysis dayanmaktadır.

Proteaz Aktivitesi belirleme Proteaz Floresan Algılama Kit kullanın

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

use-protease-fluorescent-detection-kit-to-determine-protease

Research

JoVE Journal

Biology

28.1K Görüntüleme.  Sigma Aldrich. Proteaz Floresan Algılama Kit Florimetri kullanarak proteaz aktivitesi ölçümü için tasarlanmıştır. Ayrıca eser miktarda proteaz kontaminasyon tespiti için uygundur. Yöntem FITC etiketli kazein substrat özel bir formülasyon proteolitik hydroysis dayanmaktadır.

Video: Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Plazmid DNA Qiagen Miniprep Kit kullanarak Bakteri kolonileri Arındırıcı

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Biyoloji

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

İmmünohistokimya: Parafin Bölüm Vektör Labs Vectastain ABC Seti

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, <a href="http://jove.com/index/details.stp?ID=910">click here</a>

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

Sigma&#39;nın Non-spesifik proteaz Aktivitesi Testi - Kazein bir substrat olarak

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Protein Ubikitinasyon Tespiti

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Enzim Asetat Kinaz Asetat oluşturan Faaliyet Doğrudan Algılama

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I · Cryo Kit kullanarak Fare Sperm Kriyoprezervasyon ve Kurtarma

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

Istihdam Proteaz-ve Asit-katalizli Etiketleme İş Akışları<sup&gt; 18</sup&gt; O-zenginleştirilmiş Su

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Langerhans İzolasyonu Fare Islet Saflaştırılmış Collagenase ve Nötr Proteaz bir kombinasyonu kullanılarak

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

Floresan Proteaz Koruma Kullanma Membran Protein topolojisini belirlenmesi (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...