By Alec W. M. Simpson (auth.), David G. Lambert, Richard D. Rainbow (eds.)
The legislation of intracellular Ca2+ has endured to be a strong quarter of research because the booklet of the 1st and moment variations of Calcium Signaling Protocols, and the advancements within the box have additionally, obviously, endured. With the 3rd version, specialist members discover a few of the fascinating new molecular thoughts that experience either enabled new reviews of intracellular Ca2+ rules and supplied a lot new details on strategies. created from 5 major part, the booklet covers theoretical and extremely uncomplicated suspension-based fluorimetric assays, expert dimension platforms, size of channel job, dimension of shop liberate, in addition to professional dimension ideas which come with precise probes, utilizing G-protein chimeras to strength Ca2+ signalling for screening, and genetically encoded sensors. Written within the hugely winning Methods in Molecular Biology™ sequence layout, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, effectively reproducible laboratory protocols, and tips about troubleshooting and averting identified pitfalls.
Updated and available, Calcium Signaling Protocols, 3rd Edition could be a useful reference for all these considering a circulate into the examine of intracellular Ca2+.
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Additional resources for Calcium Signaling Protocols
Truong T et al (2001) FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule. Nat Struct Biol 8:1069–1073 98. Persechini A, Lynch JA, Romoser VA (1997) Novel fluorescent indicator proteins for monitoring free intracellular Ca2+. Cell Calcium 22:209–216 99. Romoser VA, Hinkle PM, Persechini A (1997) Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence.
If Mn2+ is used as a surrogate for extracellular Ca2+, its influx into cells can be followed using fura-2 excited at 360 nm (27). Monochromator-based light sources are best for these experiments since they allow accurate excitation at the isobestic point. , >360 nm), a Ca2+-dependent decrease in fluorescence can be confused with Mn2+ entry. The relative permeability of Ca2+ influx pathways to Mn2+ may be of interest alone. (72–75). This quench technique can be used to investigate the rapid kinetics of cation entry by stopped flow fluorescence (73).
GFP-based indicators for Ca2+ have been developed that do not rely on FRET. These include the Camgaroo, Pericam, and G-CaMP probes (101–103). Tsien and colleagues (101) found that interchanging the N and C terminal portions of GFP variants around amino acids in the region between 142 and 229 (circular permutation) could yield a fluorescent protein. Surprisingly whole protein sequences could be introduced at residue 145 and the resulting chimeric protein was still fluorescent. M. Simpson 146 of EYFP (101).