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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":"<p><p>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.</p>","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
{"title":"Foliar Spray Treatment for Exogenous Application of Hormones in Maize.","authors":"Enrique Pola-Sánchez, Rodrigo Muñoz-Javier, José Alfredo Guzmán-López, María Jazmín Abraham-Juárez","doi":"10.1101/pdb.prot108621","DOIUrl":"https://doi.org/10.1101/pdb.prot108621","url":null,"abstract":"<p><p>Exogenous application of hormones in plants is a valuable technique for studying and manipulating plant growth, development, and responses to environmental stimuli. The foliar spray method is one of the most common approaches for the exogenous application of hormones in plants due to its ease of use on aerial organs (such as leaves and inflorescences) and the rapid absorption of the treated tissue, facilitating subsequent analyses. Here, we provide a protocol to implement this method in maize. The approach consists of preparing dilutions of the hormones or plant growth regulators (PGRs) of interest, usually in an aqueous solution and at low concentrations, followed by application by foliar spraying using a defined treatment regimen. Users can then evaluate effects by measuring different parameters, such as stem size, flowering time, seed production, or others. The foliar spray method can easily be scaled up and automated in greenhouse and field settings, and can be used to treat plants at all developmental stages.</p>","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":"142544160","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
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
{"title":"Apical Whorl Treatment for Exogenous Application of Hormones in Maize.","authors":"Rodrigo Muñoz-Javier, Enrique Pola-Sánchez, José Alfredo Guzmán-López, María Jazmín Abraham-Juárez","doi":"10.1101/pdb.prot108622","DOIUrl":"https://doi.org/10.1101/pdb.prot108622","url":null,"abstract":"<p><p>Plant hormones play an essential role in both development and stress responses. These organic natural compounds have critical functions in plant-related processes, including but not limited to seed development, anther formation, root elongation, and responses to abiotic and biotic stress. One way to study the impact of hormones on these processes is by external application, followed by evaluation of parameters of interest. Here, we describe one such method for the exogenous application of hormones in maize: the apical whorl treatment approach, which is well suited for evaluating the role of these compounds in reproductive stages (e.g., when the target organ is the inflorescence meristem). This method involves direct application of a hormone solution to the apical part of the plants every 2 days until the tassel emerges, which takes 15-20 days, or until the treated plants show noticeable phenotypic changes for evaluation. This method is ideal for observing effects on the apical meristem, and it may be scaled up for analyzing large numbers of plants.</p>","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":"142544158","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 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":"<p><p>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.</p>","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
{"title":"Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries.","authors":"Juan C Almagro, Mary Ann Pohl","doi":"10.1101/pdb.prot108615","DOIUrl":"https://doi.org/10.1101/pdb.prot108615","url":null,"abstract":"<p><p>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 second of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Gold Plus Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library (FL) construction, and (3) secondary library construction. The second step, described here, involves display of the PLs as single-chain variable fragment (scFv) fusions to protein pIII of the M13 phage, as well as heat shock treatment and subsequent selection of well-folded and thermostable scFvs via protein L binding, whereas unstable and defective scFvs are removed by washing steps and centrifugation. The quality of the filtration process is assessed by sequencing clones chosen at random from the FLs. These libraries, enriched with thermostable antibodies, are then ready to be used for the third and final step of the process: generation of secondary libraries.</p>","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":"142459808","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
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
{"title":"Considerations for Using Phage Display Technology in Therapeutic Antibody Drug Discovery.","authors":"Mary Ann Pohl, Juan C Almagro","doi":"10.1101/pdb.top107757","DOIUrl":"https://doi.org/10.1101/pdb.top107757","url":null,"abstract":"<p><p>Phage display is a versatile and effective platform for the identification and engineering of biologic-based therapeutics. Using standard molecular biology laboratory techniques, one can create a highly diverse and functional antibody phage-displayed library, and rapidly identify antibody fragments that bind to a target of interest with exquisite specificity and high affinity. Here, we discuss key aspects for the development of an antibody discovery strategy to harness the power of phage display technology to obtain molecules that can successfully be developed into therapeutics, including target validation, antibody design goals, and considerations for preparing and executing phage panning campaigns. Careful design and implementation of discovery campaigns-regardless of the target-provides the best chance of identifying desirable antibody fragments for further therapeutic development, so these principles can be applied to any new discovery project.</p>","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":"142459794","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":"<p><p>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 (V<sub>H</sub>) scaffold and four distinct variable light (V<sub>L</sub>) 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 V<sub>L</sub>-linker-V<sub>H</sub> 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.</p>","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
{"title":"Generation of Antibody Libraries for Phage Display: Library Reamplification.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108601","DOIUrl":"https://doi.org/10.1101/pdb.prot108601","url":null,"abstract":"<p><p>Phage display of Fab libraries enables the de novo discovery and in vitro evolution of monoclonal antibodies. Fab libraries are collections of millions to billions of different antibodies that collectively cover a large antigen or epitope binding space. To preserve the diversity of the Fab library for repeated selection campaigns, it is recommended to use the original phage from the Fab library generation rather than reamplified phage, if practically possible. This is because reamplification will bias the Fab library for clones that are expressed at higher rates. Fab-phage, however, should only be used if they have been prepared on the same day, to avoid proteolytic cleavage of the physical linkage of phenotype (phage-displayed Fab protein) and genotype (phage-encapsulated Fab DNA). Thus, in practice, reamplification of a Fab-phage library cannot usually be avoided. Here, we describe the steps for the reamplification of an original Fab-phage library prior to its selection. The protocol can also be used to reamplify Fab-phage from the third or later panning rounds when enriched clones are unlikely to be lost by reamplification biases.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459800","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
Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies. 用于生物物理和生物学研究的噬菌体展示选择 Fab 的克隆、表达和纯化。
Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108604
Matthew G Cyr, Haiyong Peng, Christoph Rader
{"title":"Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies.","authors":"Matthew G Cyr, Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108604","DOIUrl":"https://doi.org/10.1101/pdb.prot108604","url":null,"abstract":"<p><p>The antigen-binding fragment (Fab) is the ∼50-kDa monovalent arm of an antibody molecule. In the laboratory, the Fab can be produced via either enzymatic digestion or recombinant expression, and its use facilitates the accurate assessment of affinity and specificity of monoclonal antibodies. The high melting temperature of the Fab, together with its low tendency to aggregate and ready conversion to natural and nonnatural immunoglobulin (Ig) formats (without affecting antigen binding properties), have made it a preferred format for phage display, as well as a tool for accurate assessment of affinity, specificity, and developability of monoclonal antibodies. Here, we outline a strategy to clone, express, and purify human or chimeric nonhuman/human Fabs that have previously been selected by phage display. Fabs purified using this approach, which results in milligram amounts, enable a variety of downstream biophysical and biological assays that ultimately inform the success of phage display library generation and selection.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459793","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: Chimeric Rabbit/Human Fab Format. 生成用于噬菌体展示的抗体库：嵌合兔/人Fab格式。
Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108598
Haiyong Peng, Christoph Rader
{"title":"Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108598","DOIUrl":"https://doi.org/10.1101/pdb.prot108598","url":null,"abstract":"<p><p>Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires. Using phagemid family pComb3, a preferred phage display format is chimeric rabbit/human Fab, which consists of rabbit variable domains (V<sub>H</sub>, V<sub>κ</sub>, and V<sub>λ</sub>) fused to human constant domains. The human constant domains, C<sub>H</sub>1 of IgG1 and C<sub>L</sub> (C<sub>κ</sub> or C<sub>λ</sub>), not only provide established purification and detection handles but also facilitate higher expression in <i>Escherichia coli</i> compared to the corresponding rabbit constant domains. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of chimeric rabbit/human Fab libraries with randomly combined rabbit variable domains of high sequence diversity, starting from the preparation of total RNA from rabbit spleen and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 10<sup>8</sup>-10<sup>11</sup> independent chimeric rabbit/human Fab clones. As such, it can be used, for instance, for the generation of either specialized immune or large naive rabbit antibody libraries.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459798","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
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