Cold Spring Harbor protocols最新文献_第6页

Selection of Affibody Molecules Using Staphylococcal Display. 利用葡萄球菌显示筛选亲和体分子。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108401

John Löfblom, Linnea Charlotta Hjelm, Charles Dahlsson Leitao, Stefan Ståhl, Hanna Lindberg

{"title":"Selection of Affibody Molecules Using Staphylococcal Display.","authors":"John Löfblom, Linnea Charlotta Hjelm, Charles Dahlsson Leitao, Stefan Ståhl, Hanna Lindberg","doi":"10.1101/pdb.prot108401","DOIUrl":"10.1101/pdb.prot108401","url":null,"abstract":"Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for biopanning using multiple display methods. This protocol describes selection from affibody libraries using display on Staphylococcus carnosus Display of affibodies on staphylococci is very efficient and straightforward because of the single cell membrane and the use of a construct with a constitutive promoter. The workflow involves display of affibody libraries on the surface of S. carnosus cells, followed by screening and selection of binders using fluorescence-activated cell sorting (FACS). The transformation of DNA libraries into S. carnosus is less efficient and more complicated than for Escherichia coli. Because of this, staphylococcal display is suitable for affinity maturation or other protein-engineering efforts that are not dependent on very high diversity, and thus magnetic-activated cell sorting (MACS) is often not required before FACS. However, MACS is an option, and MACS procedures used for E. coli can easily be adapted for use in S. carnosus if needed.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108401"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9924641","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

Neural Stimulation during Drosophila Activity Monitor (DAM)-Based Studies of Sleep and Circadian Rhythms in Drosophila melanogaster. 基于果蝇活动监测器（DAM）的黑腹果蝇睡眠和昼夜节律研究中的神经刺激。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108180

Christopher G Vecsey, Casey Koochagian, Martin Reyes, Divya Sitaraman

{"title":"Neural Stimulation during Drosophila Activity Monitor (DAM)-Based Studies of Sleep and Circadian Rhythms in Drosophila melanogaster.","authors":"Christopher G Vecsey, Casey Koochagian, Martin Reyes, Divya Sitaraman","doi":"10.1101/pdb.prot108180","DOIUrl":"10.1101/pdb.prot108180","url":null,"abstract":"Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated by circadian rhythms, homeostatic sleep drive that builds up with wakefulness, and modifying factors such as hunger or social interactions, as well as about the biological functions of sleep. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using Drosophila activity monitors (DAMs). Here, we describe approaches for the activation of specific neurons of interest using optogenetics (involving genetic modifications that allow for light-based neuronal activation) and thermogenetics (involving genetic modifications that allow for temperature-based neuronal activation) so that researchers can evaluate the roles of those neurons in controlling rest and activity behavior. In this protocol, we describe how to set up a rig for simultaneous optogenetic or thermogenetic stimulation and activity monitoring for analysis of sleep and circadian rhythms in Drosophila, how to raise appropriate flies, and how to perform the experiment. This protocol will allow researchers to assess the causative role in the regulation of sleep and activity rhythms of any genetically tractable subset of cells.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108180"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11808860/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139711754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Analysis of Sleep and Circadian Rhythms from Drosophila Activity-Monitoring Data Using SCAMP. 利用 SCAMP 从果蝇活动监测数据中分析睡眠和昼夜节律

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108182

Christopher G Vecsey, Casey Koochagian, Maria T Porter, Gregg Roman, Divya Sitaraman

{"title":"Analysis of Sleep and Circadian Rhythms from Drosophila Activity-Monitoring Data Using SCAMP.","authors":"Christopher G Vecsey, Casey Koochagian, Maria T Porter, Gregg Roman, Divya Sitaraman","doi":"10.1101/pdb.prot108182","DOIUrl":"10.1101/pdb.prot108182","url":null,"abstract":"Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated and what biological functions it plays. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using beam break counts from Drosophila activity monitors (DAMs). The number of laboratories worldwide studying sleep in Drosophila has grown from only a few 20 years ago to hundreds today. The utility of these studies is limited by the quality of the metrics that can be extracted from the data. Many software options exist to help analyze DAM data; however, these are often expensive or have significant limitations. Therefore, we describe here a method for analyzing DAM-based data using the sleep and circadian analysis MATLAB program (SCAMP). This user-friendly software has an advantage of combining several analyses of both sleep and circadian rhythms in one package and produces graphical outputs as well as spreadsheets of the outputs for further statistical analysis. The version of SCAMP described here is also the first published software package that can analyze data from multibeam DAM5Ms, enabling determination of positional preference over time.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108182"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11552080/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139711753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Analysis of Positional Preference in Drosophila Using Multibeam Activity Monitors. 利用多波束活动监测器分析果蝇的位置偏好。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108181

Maria T Porter, Gregg Roman, Christopher G Vecsey

{"title":"Analysis of Positional Preference in Drosophila Using Multibeam Activity Monitors.","authors":"Maria T Porter, Gregg Roman, Christopher G Vecsey","doi":"10.1101/pdb.prot108181","DOIUrl":"10.1101/pdb.prot108181","url":null,"abstract":"The positional preference of an animal can be very informative regarding the choices it makes about how to interact with its environment. The fruit fly Drosophila melanogaster has been used as a robust system for examining neurobiological mechanisms underlying behavior. Fruit fly positional preference can be gathered from TriKinetics Drosophila activity monitors (DAMs), which contain four infrared beams, allowing for tracking the position of individual flies along the length of a tube. Here, we describe a method for using DAM5Ms to examine food preference. Specifically, we show an example in which circadian changes in food preference are compared between different Drosophila species. More information about the evolution of behavior can be gathered by measuring feeding preference relative to time of day. Noni, fruit from Morinda citrifolia, contains octanoic acid, a chemical toxic to many species of Drosophila D. melanogaster and D. simulans, both food generalists, show high sensitivity to octanoic acid, whereas D. sechellia, a specialist, can tolerate high concentrations. When two different food substrates are provided at each end of a tube, food preference can be inferred at various times of the day, using the sleep and circadian analysis MATLAB program (SCAMP) to extract and analyze positional data from DAM5Ms. Data gathered from these analyses can be used to compare avoidance or attraction to nutrients, tastants, or odors between species and genotypes or after specific different treatments. Additionally, such data can be examined as a function of time of day.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108181"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11467806/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139711752","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cloning of Affibody Libraries for Display Methods. 克隆用于显示方法的 Affibody 库。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108398

Stefan Ståhl, Linnea Charlotta Hjelm, Charles Dahlsson Leitao, John Löfblom, Hanna Lindberg

引用次数: 0

The Rolled Towel Method for Hormone Response Assays in Maize. 玉米激素反应测定的卷纸法

Cold Spring Harbor protocols Pub Date : 2024-10-30 DOI: 10.1101/pdb.prot108623

Clarice F Gonzales, Craig L Cowling, Dior R Kelley

引用次数: 0

Exogenous Hormone Treatments in Maize. 玉米的外源激素处理。

Cold Spring Harbor protocols Pub Date : 2024-10-30 DOI: 10.1101/pdb.top108526

José Alfredo Guzmán-López, Rodrigo Muñoz-Javier, Dior R Kelley, María Jazmín Abraham-Juárez

{"title":"Exogenous Hormone Treatments in Maize.","authors":"José Alfredo Guzmán-López, Rodrigo Muñoz-Javier, Dior R Kelley, María Jazmín Abraham-Juárez","doi":"10.1101/pdb.top108526","DOIUrl":"https://doi.org/10.1101/pdb.top108526","url":null,"abstract":"Plant hormones have key functions in plant morphology, physiology, and stress responses. Studies on the biology of hormones and their effect on plant physiology and metabolism are greatly facilitated by the exogenous application of these compounds. In general, methods for exogenous hormone application are easy and fast, and provide useful information about their effects in planta. Although hormone effects have been studied in several plant species, the used methods need to be tailored specifically to each species to get robust data. Maize is an established model for basic and applied research, and an excellent system for studying the effects of hormones on developmental and stress responses in a cereal crop. Different methods have been reported for the exogenous application of plant growth regulators in maize, including watering, spraying, immersion, and application to the apical whorl. These various methods are useful to analyze hormone responses at different developmental stages, in specific organs, and within tissues. As with all exogenous application assays, suitable experimental design and the inclusion of proper controls are critical factors in these methods, to obtain reliable and reproducible results. Here, we provide an overview of various methods for hormone exogenous application in maize, and technical considerations to get successful results.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142544159","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

Foliar Spray Treatment for Exogenous Application of Hormones in Maize. 玉米外源应用激素的叶面喷洒处理。

Cold Spring Harbor protocols Pub Date : 2024-10-30 DOI: 10.1101/pdb.prot108621

Enrique Pola-Sánchez, Rodrigo Muñoz-Javier, José Alfredo Guzmán-López, María Jazmín Abraham-Juárez

引用次数: 0

Apical Whorl Treatment for Exogenous Application of Hormones in Maize. 玉米外源应用激素的顶轮处理

Cold Spring Harbor protocols Pub Date : 2024-10-30 DOI: 10.1101/pdb.prot108622

Rodrigo Muñoz-Javier, Enrique Pola-Sánchez, José Alfredo Guzmán-López, María Jazmín Abraham-Juárez

引用次数: 0

Semisynthetic Phage Display Library Construction: Generation of Single-Chain Variable Fragment Secondary Libraries. 半合成噬菌体展示文库构建：单链可变片段二级文库的生成。

Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.prot108616

Juan C Almagro, Mary Ann Pohl

{"title":"Semisynthetic Phage Display Library Construction: Generation of Single-Chain Variable Fragment Secondary Libraries.","authors":"Juan C Almagro, Mary Ann Pohl","doi":"10.1101/pdb.prot108616","DOIUrl":"https://doi.org/10.1101/pdb.prot108616","url":null,"abstract":"Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the third and final step of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library construction, (2) filtered library (FL) construction, and (3) secondary library (SL) construction. In the third step, described here, the nucleotide sequences encoding the single-chain variable fragments (scFvs) of FLs are amplified by PCR and combined with the heavy- chain CDR3 region (HCDR3) and joining fragments (H3J) obtained from a pool of donors to maximize diversity (\"natural H3J fragments\"). These natural H3J fragments are amplified with a set of primers designed to capture >95% of the natural H3J repertoire. The resultant fragments replace the neutral H3J fragments of the FLs, resulting in the final semisynthetic secondary libraries. The quality of these libraries is assessed by sequencing clones chosen at random from the libraries, typically 96 clones. These libraries are then ready to be used for phage selections on targets of interest, providing a robust antibody discovery platform.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459809","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