Instruções de preparação O tampão de incubação, Tampão de Ensaio, e 0,6 N Solução TCA são fornecidos como ready-to-use soluções. O substrato FITC-caseína - Recomenda-se a alíquota do substrato FITC-caseína em volumes menores no momento da chegada para evitar repetidos ciclos de congelamento e descongelamento. Se o substrato FITC-caseína é submetido a repetidos ciclos de congelamento e descongelamento, um ligeiro aumento no fundo irá ocorrer, assim, diminuindo a sensibilidade. As alíquotas devem ser armazenadas a -20 ° C e protegida da luz. Cada amostra, a reação em branco, ou controle necessita de 20 mL do substrato FITC-caseína. Congelamento / descongelamento, bem como vigorosa mistura ou agitação podem fazer com que o FITC a se separar da caseína, resultando em uma maior fundo de leitura e causando menos substrato para estar disponível para a clivagem da protease. O isotiocianato de fluoresceína (FITC) Controle Solution-FITC O controle pode ser reconstituída em Tampão de Ensaio para a concentração adequada. Esta solução deve ser feita fresco e protegido da luz. Final da Solução de Controle Tripsina - Adicione 100 ml de HCl 1 mM para o frasco de tripsina, protease Control (Código do Produto T 6567). Misture rapidamente para garantir a tripsina é dissolvido. Adicionar 900 l do tampão de incubação e misture bem. Alternativamente, buffers podem ser utilizados outros se desejado para o ensaio. A concentração final de trabalho da tripsina é de 20 mg / mL. Quando estiver pronto para preparar a Solução de Controle Tripsina, combine uma alíquota da solução de tripsina ácidos com a quantidade correta do tampão de incubação (1 tripsina acidificados parte para nove partes de buffer). Armazenar a tripsina em meio ácido aumenta a estabilidade da tripsina. A solução de controle de tripsina é sensível à temperatura e não é estável em baixas concentrações. Reveja boletim técnico do kit para armazenamento e estabilidade destas soluções preparadas. Procedimento <ol><li> É mais fácil preparar controles, blanks, e amostras de uma só vez. Para cada amostra, adicionar 20 mL de tampão de incubação, 20 l de FITC-caseína de substrato, e 10 ml da amostra para um tubo de microcentrífuga. Para amostras com atividade de protease alto, diluição da amostra pode ser necessária. </li><li> Prepare amostras de controlo adequado (ver amostras de controle), adicionando 20 l de tampão de incubação, 20 l de FITC-caseína de substrato, e 10 ml da amostra de controlo a um tubo de microcentrífuga. </li><li> Prepare uma amostra em branco, adicionando 20 l de tampão de incubação, 20 l de FITC-caseína de substrato, e 10 ml de água ultrapura para um tubo de microcentrífuga. </li><li> Homogeneizar cada tubo e incubar a 37 ° C no escuro por 60 minutos. Tenha cuidado para não misturar muito vigorosamente, como turbulência excessiva pode causar fundo de fluorescência de alta e reduzir a sensibilidade do ensaio. Nota: O tempo de incubação pode ser prolongado até 24 horas para aumentar a sensibilidade. Tenha cuidado para não exceder as 24 horas como o FITC-caseína podem começar a se degradar, levando ao fundo de fluorescência de alta. </li><li> Após a incubação adicionar 150 mL da solução de ácido 0,6 N tricloroacético a cada tubo de microcentrífuga. TCA é muito corrosivo e deve ser tratado enquanto usava o equipamento de protecção adequado. </li><li> Homogeneizar e incubar a 37 ° C no escuro por 30 minutos. </li><li> Centrifugue os tubos durante 10 minutos a 10.000 x g. O sobrenadante contém o ácido solúvel, FITC fragmentos rotulados e é utilizado para a medição de fluorescência. </li></ol> Medidas de fluorescência Estes métodos podem ser ampliados ou reduzidos de acordo com os requisitos da instrumentação disponível. Para comparação com uma curva padrão preparada com as amostras de controlo adequadas, subtrair a leitura de fluorescência da amostra em branco (FLUblank) do valor de cada amostra (FLUtest). Cubetas <ol><li> Pipetar 10 mL do sobrenadante (passo 7) e 1 ml de Tampão de Ensaio numa cuvete adequado e misture delicadamente. Nota: A solução de Tampão de Ensaio sobrenadante e podem ser armazenadas no escuro a 2-8 ° C por até 24 horas antes de medir a fluorescência. </li><li> Registrar a intensidade de fluorescência com excitação em 485 nm e monitoramento do comprimento de onda de emissão de 535 nm. </li></ol> Placas com vários poços <ol><li> Pipetar 10 mL do sobrenadante Nota (passo 7) e 1 ml de Tampão de Ensaio em um tubo ou frasco adequado e misture delicadamente: A solução de Tampão de Ensaio sobrenadante e podem ser armazenadas no escuro a 2-8 ° C por até a 24 horas antes de medir a fluorescência. </li><li> Transferência de 200 mL para um poço de uma placa de 96 preto também. Registrar a intensidade de fluorescência com excitação em 485 nm e monitoramento do comprimento de onda de emissão de 535 nm. Ou </li></ol><ol><li> Pipetar 2 mL do sobrenadante (etapa 7) e 200 mL do B Assayuffer em um poço de uma placa de 96 preto também. Nota: A solução de Tampão de Ensaio sobrenadante e podem ser armazenadas no escuro a 2-8 ° C por até 24 horas antes de medir a fluorescência. </li><li> Registrar a intensidade de fluorescência com excitação em 485 nm e monitoramento do comprimento de onda de emissão de 535 nm. </li></ol> As amostras de controle A Solução de Controle de tripsina pode ser usado para confirmar o teste está funcionando adequadamente, para determinar o limite de detecção, ou criar uma curva de norma geral. Para o ensaio de uma protease, diferentes específico, recomenda-se para preparar uma solução de controle que contém a protease específica no buffer de incubação apropriado. O limite de detecção do ensaio é a quantidade de protease, que produz uma leitura significativa de fluorescência acima do valor obtido com a amostra em branco. O limite de detecção irá variar dependendo da sensibilidade dos instrumentos. Diluições seriadas da Solução de Controle Tripsina pode ser usado para gerar as soluções controle. A leitura igual a 120% do valor obtido com a amostra em branco é considerado significativo. Rotineiramente, um limite de detecção de 5 ng de tripsina foi obtida com este procedimento. Recomenda-se que pelo menos um 5 ng controle de tripsina ser executado a cada ensaio. A solução de controle de tripsina com uma concentração de 0,5 mg / ml resultaria na desejado 5 ng de tripsina no ensaio. A diluição de 40 vezes da Solução de Controle de tripsina (20 mcg / ml) resulta em uma solução de controle de 0,5 ug / ml, ou seja, uma parte da solução de tripsina de controle para 39 partes de tampão de incubação. <img src="/files/ftp_upload/1514/figure1.jpg" alt="Figura 1" /> Figura 1 (curva padrão de atividade de tripsina): A Solução de Controle Tripsina (20 mcg / ml) também pode ser usado para gerar uma curva padrão, fazendo diluições em série (ver Figura 1). A leitura de fluorescência de cada amostra de controlo foi corrigido subtraindo-se a leitura de fluorescência da amostra em branco (FLUblank) do valor de cada amostra de controlo (FLUcontrol). Curva padrão típico de tripsina (Product Code T 6567) utilizando este kit e amostras de controle que varia de 0,15 mg / ml (1,5 ng) a 2,5 mg / ml (25 ng). Esta curva foi gerada através do procedimento descrito, com um tempo de incubação de 30 minutos para a etapa 4. Medições de fluorescência foram feitos em uma placa com vários poços. Isotiocianato de fluoresceína é fornecido como um controle de calibração de instrumentos possíveis (ver instruções do fabricante apropriado) ou determinação da faixa de linearidade do sinal de FITC.

<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>

O autor é um funcionário da Sigma Aldrich que produz reagentes e ferramentas utilizadas neste artigo.

Temos mostrado apenas um ensaio exemplo usando o Kit de Detecção de Protease fluorescente para detectar atividade de protease em uma amostra biológica. Este kit foi otimizado para detectar uma gama diversificada de proteases encontrados em aplicações fisiológicas. Ele é adequado para a detecção de cisteína, serina, metalo e proteases aspártico, no entanto, modificações podem ser necessários para detectar alguns proteases específicas.

<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

O Kit de Detecção de Protease fluorescente fornece ready-to-use reagentes para a detecção da presença de atividade da protease. Este ensaio simples para detectar atividade de protease utiliza caseína marcado com isotiocianato de fluoresceína (FITC) como substrato. Protease atividade resulta na clivagem do substrato de caseína FITC-rotulados em fragmentos menores, que não precipitam em meio ácido. Após a incubação da amostra protease e substrato, a reação é acidificada com a adição de ácido tricloroacético (TCA). A mistura é então centrifugado com o substrato formando um pellet não digerido e as menores, fragmentos de ácido solúvel remanescente em solução. O sobrenadante é neutralizado ea fluorescência do FITC-rotulados fragmentos é medido. O procedimento descrito kit detecta o controle protease tripsina em uma concentração de aproximadamente 0,5 mcg / ml (5 ng de tripsina adicionado ao ensaio). Esta sensibilidade pode ser aumentada com um maior tempo de incubação, até 24 horas. O ensaio é realizado em tubos de microcentrífuga e procedimentos são fornecidos para a detecção de fluorescência usando tinas ou placas de vários poços.

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

O Kit de Detecção de Protease fluorescente é projetado para a medição da atividade da protease usando fluorometria. Também é adequado para a detecção de vestígios de contaminação protease. O método é baseado no hydroysis proteolítica de uma formulação de um substrato de caseína FITC-rotulados.

Uso do Kit de Detecção de Protease Fluorescente para determinar a atividade de protease

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

28.0K visualizações.  Sigma Aldrich. O Kit de Detecção de Protease fluorescente é projetado para a medição da atividade da protease usando fluorometria. Também é adequado para a detecção de vestígios de contaminação protease. O método é baseado no hydroysis proteolítica de uma formulação de um substrato de caseína FITC-rotulados.

Vídeo: 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.

Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

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

Biologia

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.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

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>

Sigma&#039;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.

Não-específicas da Sigma ensaio de atividade de protease - A caseína como substrato

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 ...

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.

Detecção direta da Atividade Acetato de formação do Acetato Quinase Enzima

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

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.

Protease e catalisada por ácido Workflows Labeling Empregando<sup&gt; 18</sup&gt; O-enriquecido Água

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.

Rato Ilhéu de Langerhans isolamento usando uma combinação de colagenase purificada e Protease Neutra

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.

Determinando Membrane Topology Protein Usando Fluorescência 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 ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

O uso de<em&gt; Ex Vivo</em&gt; Imagem de órgãos inteiros e Histologia Tissue quantitativa para determinar o Bio-distribuição das moléculas fluorescente etiquetado

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

O ensaio de sequestro de lactato desidrogenase — Um método simples e confiável para determinar a atividade de Autophagic de sequestro em massa em células de mamíferos

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical diagnostics. It can be used to characterize various biological processes or pathologies, such as wound-healing, immune response, tissue rejection, and tissue-biomaterial interactions. However, the visualization and quantification of multiple antigens (especially for immune cells) in a single tissue section using conventional immunohistochemical (IHC) staining remains unsatisfactory. Hence, multiplexed technologies were introduced in recent years to identify multiple biological markers in a single tissue sample or an ensemble of different tissue samples. 
These technologies can be especially useful in differentiating the changes in immune cell-to-cell interactions within the endometrium between fertile women and women with recurrent miscarriages during implantation. This paper describes a detailed protocol for multiplexed fluorescence IHC staining to investigate the density and clustering of four major immune cell types simultaneously in precisely timed endometrial specimens during embryo implantation. The method includes sample preparation, multiplex optimization with markers for immune cell subtypes, and the scanning of the slides, followed by data analysis, with specific reference to detecting endometrial immune cells. 
Using this method, the density and clustering of four major immune cell types in the endometrium can be simultaneously analyzed in a single tissue section. In addition, this paper will discuss the critical factors and troubleshooting to overcome possible fluorophore interference between the fluorescent probes being applied. Importantly, the results from this multiplex staining technique can help provide an in-depth understanding of the immunologic interaction and regulation during embryo implantation.

Despite the advancements in multiplex immunohistochemistry and multispectral imaging, characterizing the density and clustering of major immune cells simultaneously in the endometrium remains a challenge. This paper describes a detailed multiplex staining protocol and imaging for the simultaneous localization of four immune cell types in the endometrium.

Coloração imunohistoquímica fluorescente multiplexada de quatro tipos de células imunes endometrial em aborto recorrente

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical ...