Cold Spring Harbor protocols最新文献_第6页

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

Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries. 半合成噬菌体展示文库构建：过滤文库的生成。

Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.prot108615

Juan C Almagro, Mary Ann Pohl

引用次数: 0

Considerations for Using Phage Display Technology in Therapeutic Antibody Drug Discovery. 在治疗性抗体药物研发中使用噬菌体展示技术的注意事项。

Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.top107757

Mary Ann Pohl, Juan C Almagro

引用次数: 0

Maize Abiotic Stress Treatments in Controlled Environments. 受控环境中的玉米非生物胁迫处理。

Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.prot108620

Katherine M Murphy, Anna L Casto, Leonardo Chavez, Leonardo W Lima, Alejandra Quiñones, Malia A Gehan, Cory D Hirsch

{"title":"Maize Abiotic Stress Treatments in Controlled Environments.","authors":"Katherine M Murphy, Anna L Casto, Leonardo Chavez, Leonardo W Lima, Alejandra Quiñones, Malia A Gehan, Cory D Hirsch","doi":"10.1101/pdb.prot108620","DOIUrl":"https://doi.org/10.1101/pdb.prot108620","url":null,"abstract":"Maize (Zea mays) is one of the world's most important crops, providing food for humans and livestock and serving as a bioenergy source. Climate change and the resulting abiotic stressors in the field reduce crop yields, threatening food security and the global economy. Water deficit (i.e., drought), heat, and insufficient nutrients (e.g., nitrogen and phosphorus) are major environmental stressors that affect maize yields, and impact growth and development at all stages of the plant life cycle. Understanding the biological processes underlying these responses in maize has the potential to increase yields in the face of abiotic stress. Optimizing individual or combined abiotic stress treatments in controlled environments reduces potential noise in data collection that can be present under less controlled growth conditions. Here, we describe methods and conditions for controlled abiotic stress treatments and associated controls during early vegetative growth of maize, conducted in greenhouses or growth chambers. This includes the environmental conditions, equipment, soil preparation, and intensity and duration of heat, drought, nitrogen deficiency, and phosphorous deficiency. Controlled experiments at early growth stages are informative for future in-field studies that require greater labor and inputs, saving researchers time and growing space, and thus research funds, before testing plants across later stages of development. We suggest that stress treatments be severe enough to result in a measurable phenotype, but not so severe that all plants die prior to sample collection. This protocol is designed to set important standards for replicable research in maize.","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":"142459803","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

Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries. 半合成噬菌体展示文库构建：多样化单链可变片段的设计与合成以及初级文库的生成。

Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.prot108614

Juan C Almagro, Mary Ann Pohl

{"title":"Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries.","authors":"Juan C Almagro, Mary Ann Pohl","doi":"10.1101/pdb.prot108614","DOIUrl":"https://doi.org/10.1101/pdb.prot108614","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 first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (VH) scaffold and four distinct variable light (VL) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a VL-linker-VH orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.","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":"142459807","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

Generation of Antibody Libraries for Phage Display: Library Reamplification. 生成用于噬菌体展示的抗体库：文库再扩增

Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108601

Haiyong Peng, Christoph Rader

引用次数: 0