By Peter Satir, Søren Tvorup Christensen
This quantity is the results of an explosion of molecular-based examine on Cilia, which started with the invention of the universality of intraflagellar shipping (IFT) and ciliary genomics/proteomics. The chapters during this publication conceal themes comparable to: excessive solution imaging and sensible characterization of sensory and first cilia in mammalian cells and zebrafish, tips on how to examine ciliary-mediated chemoresponse in Paramecium, and techniques to check centrosomes and cilia in C. elegans and Drosophila. Written within the hugely profitable tools in Molecular Biology sequence structure, chapters contain advent to their respective subject matters, lists of the required fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and pointers on troubleshooting and fending off identified pitfalls.
Practical and state of the art, Cilia: tools and Protocols is wide and covers motile, sensory, and first cells. it's a important source to an individual attracted to coming into the sphere of ciliary biology utilizing version organisms, together with flagellate algae, ciliates, planaria, nematodes, bugs, zebrafish, and mammalian cells.
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Extra info for Cilia: Methods and Protocols
3. Ethanol (70 and 96 %). 4. 100 % methanol. 23 Centrosome-Targeting of ASH Domain Proteins 5. PBS. 6. 4 % paraformaldehyde (PFA) solubilized in PBS. 7. 2 % (v/v) Triton X-100 and 1 % (v/v) BSA in PBS. 8. Blocking buffer: 2 % (w/v) bovine serum albumin (BSA) in PBS. 9. Primary antibodies (dilutions in parenthesis): goat anti-Myc (1:1000) from Abcam; rabbit anti-ARL13B (1:1000) from Protein Tech; mouse anti-p150Glued (1:500) from BD Transduction Laboratories; mouse anti-FLAG (1:1000) from Sigma.
6 Preparation of Coverslips for Immunofluorescence Microscopy For NT2 cell growth, 12 mm coverslips for immunofluorescence microscopy analysis are treated with acid prior to growing cells on them (see Note 2). 40 Rania Ghossoub et al. 1. Incubate coverslips in 32 % HCl for 60 min. Mix frequently by swirling bottle around. This step should be performed in the fume hood. 2. Discard HCl (can be saved and reused) and add water, continue working in the fume hood. Swirl and discard. You can now move away from the hood for washing coverslips 15 times in ddH2O.
Mol Cell Biol 29(11):3134–3150. 01663-08 Januschke J, Reina J, Llamazares S, Bertran T, Rossi F, Roig J, Gonzalez C (2013) Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts. Nat Cell Biol 15(3):241–248. 1038/ncb2671 Pazour GJ, Agrin N, Leszyk J, Witman GB (2005) Proteomic analysis of a eukaryotic cilium. J Cell Biol 170:103–113 Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson A, Kampf C, Sjostedt E, Asplund A, Olsson I, Edlund K, Lundberg E, Navani S, Szigyarto CA, Odeberg J, Djureinovic D, Takanen JO, Hober S, Alm T, Edqvist PH, Berling H, Tegel H, Mulder J, Rockberg J, Nilsson P, Schwenk JM, Hamsten M, von Feilitzen K, Forsberg M, Persson L, Johansson F, Zwahlen M, von Heijne G, Nielsen J, Ponten F (2015) Proteomics.
Cilia: Methods and Protocols by Peter Satir, Søren Tvorup Christensen