By Enrique López-Juez, Alison Hills (auth.), R. Paul Jarvis (eds.)
Chloroplasts are crucial for the survival and flourishing of existence on the earth. through the years, chloroplast biology has been studied in a number of various organisms, resulting in the numerous drawback that findings that have been made through the use of diverse experimental structures or species weren't regularly at once cross-comparable. The fairly contemporary adoption of Arabidopsis thaliana because the version organism of selection for plant technology examine, around the globe, has resulted in its emergence as a pre-eminent approach for examine on chloroplasts and different different types of plastid. In Chloroplast learn in Arabidopsis: equipment and Protocols, specialist researchers compile probably the most very important, smooth ideas and techniques for chloroplast study, with the unifying topic of Arabidopsis because the version process. Volume I explores themes similar to genetics, cytology, in vivo research, gene expression, and protein accumulation, in addition to protein delivery, localization, and topology. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and keeping off recognized pitfalls.
Authoritative and handy, Chloroplast study in Arabidopsis: equipment and Protocols serves as an amazing reference for all researchers with a common curiosity in chloroplasts, plastids, or similar processes.
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Extra resources for Chloroplast Research in Arabidopsis: Methods and Protocols, Volume I
For convenient dual labeling, the primary antibodies should be raised in different species and the secondary antibody conjugates should have minimal cross-reactivity. , neither the primary nor secondary antibodies should be mixed together, and thorough washing should be performed after each incubation. Controls for double labeling should include reverse order of labeling and omission of one of the primary antibodies to check for cross-reactivity of the reagents. Secondary antibody conjugates with traditional dyes such as fluorescein and rhodamine are outdated; these dyes have been surpassed by newer, much brighter and more photostable dyes.
1. 5-mL Eppendorf tube; do this in a fume hood, since glutaraldehyde vapour is toxic. If several samples are to be processed, then 1 mL of glutaraldehyde in each of several Eppendorf tubes is required. Harvest leaves or pieces of leaf tissue from growing Arabidopsis plants in which chloroplasts are to be analysed, submerge them in the glutaraldehyde solution, and close the lid. 5 cm2 can be fixed entirely but larger leaves are best cut into strips 1–5 mm wide with a scalpel or razor blade. It is important that all tissues are submerged in the fixative.
For cryosectioning, the tissue may be infiltrated with a cryoprotectant, such as sucrose, to minimize ice crystal formation, and then frozen and sectioned using a cryostat (8–10). Such sections generally exhibit very good preservation of antigenicity, but frozen plant tissue is difficult to section and the resulting sections are often damaged. Wax embedding permits convenient sectioning with good structural preservation of plant tissues. The classical paraffin wax embedding medium requires relatively high temperatures for embedding (~60°C), which causes reduced or sometimes complete loss of antigenicity.