Protocols used in Molecular Biology最新文献

Osteosarcoma Cell Culture and Maintenance to Detect the Apoptotic Effect of Some Promising Compounds by Potent Markers viz. DNA 骨肉瘤细胞培养与维持:利用DNA等有效标记检测一些有希望的化合物的凋亡作用

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010014

引用次数: 0

Protocols for the Detection and Proteome Analysis of the Yellow Mosaic Virus Infected Soyabean Leaves 大豆叶片黄花叶病毒检测及蛋白质组学分析方案

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010009

引用次数: 0

RNA Isolation Protocol from Cells and Tissues 细胞和组织的RNA分离方案

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010005

{"title":"RNA Isolation Protocol from Cells and Tissues","authors":"","doi":"10.2174/9789811439315120010005","DOIUrl":"https://doi.org/10.2174/9789811439315120010005","url":null,"abstract":"The preparation of intact ribonucleic acid is difficult because of the action of\u0000nucleases, which are liberated upon tissue homogenisation. In many cells, high\u0000concentrations of the ribonucleases are reserved in the secretory granules and upon\u0000disruption of the cell, they get mixed with the RNA and lead to its degradation.\u0000Guanidinium chloride and thiocyanate are potent chaotropic agents that reduce\u0000hydrophobic interactions and disrupt protein tertiary structures, disassociate proteinnucleic\u0000acid complexes and disintegrate cellular structures. Guanidinium thiocyanate is\u0000especially strong protein denaturant because both the cation and anion disrupt the\u0000hydrophobic bonds between the amino acid side chains. RNA usually binds to proteins\u0000within a cell and this agent disassociates the nucleoprotein complex, without disrupting\u0000RNA structure. Thus RNA can be obtained by using these agents, after homogenisation\u0000and low-speed centrifugation and precipitated with ethanol. The protocol below\u0000explains the stepwise isolation of total RNA from cells and tissues using TRIzol\u0000reagent which is the mono-phasic solution of phenol and guanidine thiocyanate.","PeriodicalId":179247,"journal":{"name":"Protocols used in Molecular Biology","volume":"80 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2020-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"128748650","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Dictyostelium Discoideum: Live Cell Imaging in Changing Perspective 盘状骨:变化视角下的活细胞成像

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010016

{"title":"Dictyostelium Discoideum: Live Cell Imaging in Changing Perspective","authors":"","doi":"10.2174/9789811439315120010016","DOIUrl":"https://doi.org/10.2174/9789811439315120010016","url":null,"abstract":"The advent of advanced microscopes; during microscope evolution from\u0000simple microscopes to confocal and live cell microscope; having digital imaging\u0000facility revolutionized our view for the living cells. In the protein localization study,\u0000fluorescent proteins are tagged at amino or carboxyl (preferably) terminal of desired\u0000protein for live cell study. These live cell studies improved our understanding of\u0000protein dynamics and understanding its role in biological regulation. The mutational\u0000variants of fluorescent tags (GFP, RFP); can be used with different protein; which will\u0000efficiently use UV-Visible to Far Red light spectrum; without overlapping of excitation\u0000and emission spectrum. Further, various cell organelle (Lysosome, Golgi bodies,\u0000Endoplasmic Reticulum, Mitochondria, Nucleus) trackers; improved our live cell\u0000localization studies in the wide non-overlapping UV-Visible spectrum.This chapter\u0000gives an overview for live cell protein localization study in mitotically active,\u0000unicellular stage of Dictyostelium discoideum. This evolutionary cutting edge organism\u0000had both unicellular as well as multicellular stages during its life cycle. This chapter\u0000will provide the design of fusion of fluorescent tag to the specific gene and its live cell\u0000localization. Further, it will cover; transformation of the unicellular organism; drug\u0000based selection; sample preparation with nuclear, mitochondrial localization markers\u0000(trackers) and live cell localization study on live cell-confocal microscope setup. It will\u0000also have a glimpse of the design of fusion protein with an aspect of advantage and\u0000disadvantages.","PeriodicalId":179247,"journal":{"name":"Protocols used in Molecular Biology","volume":"27 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2020-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134228181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Immunohistochemistry as an Important Technique in Experimental and Clinical Practices 免疫组织化学在实验和临床中的重要作用

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010008

{"title":"Immunohistochemistry as an Important Technique in Experimental and Clinical Practices","authors":"","doi":"10.2174/9789811439315120010008","DOIUrl":"https://doi.org/10.2174/9789811439315120010008","url":null,"abstract":"Immunohistochemistry (IHC) is a well-known technique in the field of\u0000biological and medical sciences. This technique is based on the principle of antigenantibody\u0000interaction and is used for identification of cellular or tissue constituents, i.e.,\u0000an antigen by using a specific antibody. The binding of an antibody to an antigen is\u0000confirmed either by labelled primary antibody itself or by using secondary labelling\u0000method such as fluorescence labelled antibody. Such interactions give information\u0000about the cellular process occurring inside the cell. In last few years, huge amount of\u0000data have been generated using IHC. Furthermore, adequate knowledge of this\u0000technique is required for the optimum result and its reproducibility. The detailed\u0000information about the tissue section, antigen retrieval (AR), increased sensitivity of the\u0000detection systems and proper standardization are the key points for this technique. This\u0000protocol will address overview of the technique, tissue preparation, microtome, antigen\u0000retrieval, antibodies and antigen fixation, detection methods, background reduction and\u0000trouble shootings.","PeriodicalId":179247,"journal":{"name":"Protocols used in Molecular Biology","volume":"15 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2020-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121061892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Molecular Techniques for Genotyping 基因分型的分子技术

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010011

{"title":"Molecular Techniques for Genotyping","authors":"","doi":"10.2174/9789811439315120010011","DOIUrl":"https://doi.org/10.2174/9789811439315120010011","url":null,"abstract":"Genotyping is a process of determining the genetic constituent/genetic\u0000makeup “genotype” of an organism by examining the individual DNA sequence and\u0000comparing to a reference or other individual sequence. It helps the researchers to\u0000explore the genetic constitution, genetic linkages or variations like Single Nucleotide\u0000Polymorphisms (SNP) or multi-nucleotide changes in DNA. Identification of\u0000genotypes is also useful for determining their role in phenotypic expressions.\u0000Genotyping is an essential tool for researchers to find out disease-associated genes and\u0000gene variants. Genotype determined can also be used for the identification of\u0000susceptibility and prognosis for any disease and to find out responders/non-responders\u0000for a specific treatment, thus leading the way towards personalized medicine. Several\u0000molecular techniques have provided swift, reliable and accurate ways for determining\u0000genotypes. The process of genotyping involves molecular techniques like isolation and\u0000quantification of genomic DNA, visualization of DNA on agarose/polyacrylamide gel\u0000using electrophoresis, polymerase chain reaction (PCR), restriction fragment length\u0000polymorphism (RFLP), random amplified polymorphic detection (RAPD) of genomic\u0000DNA, amplified fragment length polymorphism (AFLP), sequencing, allele-specific\u0000oligonucleotide (ASO) probes, microarrays etc. The present chapter will describe the\u0000protocols for different molecular techniques that are used to determine genotypes.","PeriodicalId":179247,"journal":{"name":"Protocols used in Molecular Biology","volume":"125 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2020-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131884737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

2D-DIGE A Powerful Tool for Proteome Analysis 2D-DIGE:蛋白质组分析的强大工具

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010010

{"title":"2D-DIGE A Powerful Tool for Proteome Analysis","authors":"","doi":"10.2174/9789811439315120010010","DOIUrl":"https://doi.org/10.2174/9789811439315120010010","url":null,"abstract":"In the recent past, two dimensional gel electrophoresis has emerged as a\u0000powerful molecular biology tool for the comparative expression profiling of complex\u0000protein sample. It involves the separation as well as the resolution of diverse proteins\u0000sample on the basis of isoelectric points and molecular mass of protein in two\u0000dimension ways. In this way, it reflects the view of overall proteome status including\u0000differentiation in protein expression levels, post-translational modifications etc.\u0000Moreover, this allows the identification of novel biological signatures, which may give\u0000a particular identity of pathological background to cells or tissues associated with\u0000various types of cancers and neurological disorders. Therefore, by utilizing such tools,\u0000one can clearly investigate and compare the effects of particular drugs on cells of\u0000tissues and also one can analyze the effects of disease on the basis of variations in\u0000protein expression profile at broad spectrum. Recently, to get more error-less and\u0000accurate proteome profile, conventional 2-D gel electrophoresis has been enhanced\u0000with the inclusion of different types of protein labeling dyes which enables a more\u0000comparative analysis of diverse protein sample in a single 2-D gel. In this advanced\u0000technique (2-D-DIGE), protein samples are labeled with three different types of\u0000CyDyes (Cy2, Cy3, and Cy5) separately and combined and further resolved on the\u0000same gel. This will facilitate the more accurate spot matching on a single gel platform\u0000and will also minimize the experimental variations as commonly reported in the\u0000conventional 2D-gel electrophoresis. Therefore, in the present proteomic research era,\u00002D-DIGE has proved to be an extremely powerful tool with great sensitivity for up to\u0000125 ng of proteins in clinical research volubility especially, neurological and cancer\u0000related disorders.","PeriodicalId":179247,"journal":{"name":"Protocols used in Molecular Biology","volume":"23 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2020-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121895133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Sodium Bisulfite Conversion of Human Genome for DNA Methylation Studies 亚硫酸氢钠转化人类基因组用于DNA甲基化研究

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010012

{"title":"Sodium Bisulfite Conversion of Human Genome for DNA Methylation Studies","authors":"","doi":"10.2174/9789811439315120010012","DOIUrl":"https://doi.org/10.2174/9789811439315120010012","url":null,"abstract":"The regulation of transcription and translation of a gene under a given\u0000environment is dependent on several factors and epigenetics is one such factor,\u0000responsible for the differential expression of several genes in health and in various\u0000diseases. DNA methylation, an important epigenetics mechanism has been shown to\u0000play a vital role in numerous cellular processes, and the abnormal patterns of\u0000methylation have been linked to the number of human diseases. CpG islands, a short\u0000stretch of DNA enriched with CpG sites in the 5’ end of a gene, although remains\u0000unmethylated but tends to methylate aberrantly upon certain environmental exposures.\u0000The methylation of the promoter region bearing transcriptional start sites of those genes\u0000that encodes tumor suppressors such as tumor protein p53, retinoblastoma-associated\u0000protein 1, tumor protein p16, breast cancer 1 and many more result in the reduced\u0000expression of these genes and have been implicated in a large number of cancers like\u0000retinoblastoma, colon, lung and ovarian. A growing number of human diseases have\u0000been found to be associated with the aberrant DNA methylation. Hence, a deep insight\u0000into the individual’s epigenetic profile is the need of the hour. Several approaches have\u0000been developed to map DNA methylation patterns genome-wide. Some of these\u0000approaches include enzymatic digestion with methylation-sensitive restriction\u0000enzymes, the capture of 5-mC by methylated DNA-binding proteins followed by nextgeneration\u0000sequencing and methyl-DNA immunoprecipitation followed by sequencing\u0000of precipitated fragments. However, this chapter is going to describe the most\u0000recommended method for studying DNA methylation pattern, the method based on\u0000bisulfite sequencing. The bisulfite treatment of DNA converts unmethylated cytosine(s)\u0000to uracil(s), which are subsequently amplified as Ts by PCR. Hence, the bisulfitetreated\u0000DNA has mutations specifically at unmethylated Cs that can be mapped by\u0000Next-Generation sequencing.","PeriodicalId":179247,"journal":{"name":"Protocols used in Molecular Biology","volume":"29 5-6","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2020-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121002423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Chromatin Immunoprecipitation (ChIP) 染色质免疫沉淀(ChIP)

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010013

Bingnan Gu

{"title":"Chromatin Immunoprecipitation (ChIP)","authors":"Bingnan Gu","doi":"10.2174/9789811439315120010013","DOIUrl":"https://doi.org/10.2174/9789811439315120010013","url":null,"abstract":"Chromatin immunoprecipitation or ChIP is an excellent method of\u0000investigation of the specific protein interaction and its altered forms with DNA region.\u0000These interactions have a significant role in various cellular processes such as\u0000replication, transcription, DNA damage repair, genome stability, gene regulation and\u0000segregation at mitosis. This technique is therefore giving us power to study a variety of\u0000cellular mechanisms inside the cell in terms of protein-DNA interaction. As the name\u0000Chromatin immunoprecipitation suggests this method utilizes chromatin preparation\u0000from cells to selectively immune-precipitate the protein of interest to identify DNA\u0000sequence associated with it. Chromatin is an organized structure of eukaryotic DNA\u0000which contains double-stranded DNA wrapped around nucleosomes. ChIP has been\u0000extensively used to depict transcription factors, variants of histone, chromatin\u0000modifying enzymes, post-translational modification of histone on the genome. In the\u0000classical ChIP method, protein and DNA is irreversibly cross-linked by UV exposure\u0000followed by immunoprecipitation with specific antibodies, protein-DNA complex is\u0000then purified, treated with proteases and then analysis is done by the method of\u0000Southern blot or dot blot using a radio-labelled probe derived from the cloned DNA\u0000fragment of interest. Further, it was modified by using formaldehyde for reversible\u0000cross-linking of protein-DNA complex and polymerase chain reaction for the detection\u0000of fragments of precipitated DNA. ChIP is a cumbersome procedure to perform and\u0000present many limitations, for example it requires many cells. Therefore, many\u0000modifications and variations, have also developed with the time which enables us to\u0000simplify the procedure and widen its range of applications. This chapter provides a\u0000brief method for Chromatin immunoprecipitation (ChIP) and its applications.","PeriodicalId":179247,"journal":{"name":"Protocols used in Molecular Biology","volume":"228 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2020-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131773193","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Modified Western Blot Protocol for Enhanced Sensitivity in the Detection of a Tissue Protein 提高组织蛋白检测灵敏度的改进Western Blot方案

Protocols used in Molecular Biology Pub Date : 2020-01-22 DOI: 10.2174/9789811439315120010007

{"title":"A Modified Western Blot Protocol for Enhanced Sensitivity in the Detection of a Tissue Protein","authors":"","doi":"10.2174/9789811439315120010007","DOIUrl":"https://doi.org/10.2174/9789811439315120010007","url":null,"abstract":"Western blots (WB) are designed to investigate protein levels and their\u0000patterns of modification in homogenized tissue samples. Although, Western blots are\u0000quantifiable, unlike immunohistochemistry, cellular integrity is lost. The availability of\u0000antibodies against the protein and their patterns of modification of interest form the\u0000basis of both Western blots and Immunohistochemistry. Antibodies can also be\u0000directed not only against proteins but against chemical modifications of the proteins\u0000too, such as phosphorylation and glycosylation of specific amino acid residues. In\u0000Western blotting, the proteins in the sample are denatured, size-separated on a\u0000denaturing acrylamide gel, and transferred to a nylon membrane. Antibody paratopes\u0000can then bind to the antigenic epitope in the protein present on the nylon membrane.\u0000Thus, with the help of a chemiluminescent assay system that darkens X-ray films, the\u0000resulting antibody-antigen complex can be visualized. Because of the ubiquitous and\u0000relatively inexpensive availability of WB equipment, the quality of WB in publications\u0000and following analysis and investigation of the data can be variable, possibly resulting\u0000in forged conclusions. This may be because of the poor laboratory technique and/or\u0000lack of understanding of the significant steps involved in WB and what quality control\u0000procedures should be followed to ensure effective data generation. The present book\u0000chapter focuses on providing a detailed description and critique of WB procedures and\u0000technicalities, from sample collection through preparation, blotting, and detection, to\u0000examination of the data collected. We aim to provide the reader with the improved\u0000expertise to decisively carry out, assess, and troubleshoot the WB process, in order to\u0000produce reproducible and reliable blots.","PeriodicalId":179247,"journal":{"name":"Protocols used in Molecular Biology","volume":"8 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2020-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131882053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0