By Richard Nuccitelli, Leslie Wilson, Paul T. Matsudaira
A Practical consultant to the examine of Calcium in dwelling Cells describes renowned strategies besides valuable do's and don't's and laptop courses. the quantity permits investigators to guage confocal pictures, use the most recent dyes, and layout Calcium buffers applicable to their examine wishes. This e-book is designed for laboratory use by way of graduate scholars, technicians, and researchers in lots of disciplines, starting from molecular to mobile degrees of research. Key good points * Describes concepts for detection of [Ca2+]I: Ca2+ - delicate microelectrodes * Fluorescent dyes * Luminescent proteins * contains thoughts for perturbing intracellular Ca2+ * Covers precise method plus difficulties and pitfalls of every method * incorporates a sensible advisor to getting ready Ca2+ buffers with an easy-to-use machine software * colour plates illustrate options corresponding to * Confocal ratio-imaging * Use of aequorin
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Extra info for A Practical Guide to the Study of Calcium in Living Cells
Ac’ta 839, 287-299. Smith, P. , Berger, R. , and Podolsky, R . J . (1977). Stopped-flow study of the rate of calcium binding by EGTA. Biophys. J . 17, 159a. Taylor, R . B . Trimble. C.. Valdes. J. J . Wayner. M. , and Chambers. J . P. (1992). Determination of free calcium. Brain Rrs. Bull. 29,499-501. Trosper. T. , and Philipson. K. D. (1984). Stimulatory effect of calcium chelators on Na+-Ca’+ exchange in cardiac sarcolemmal vesicles. Cell Cdcirrm 5, 21 1-222. Tsien, R . Y . (1980). New calcium indicators and buffers with high selectivity against magnesium and protons: Design.
111. DM-Nitrophen A. Chemical Properties A different strategy for releasing Ca2+ involves attaching a 2-nitrobenzyl group to one of the chelating amines of ethylenediaminetetraacetic acid (EDTA) to form the photosensitive chelator DM-nitrophen (Ellis-Davies and Kaplan, 1988; Kaplan and Ellis-Davies, 1988). , 1992) to form iminodiacetic acid and a 2nitrosoacetophenone derivative within less than 180 psec. The reaction is shown in Fig. 3. 25 m M at an ionic strength of 150 mM (Kaplan and Ellis-Davies, 1988; Neher and Zucker, 1993).
Environmental conditions (pH, ionic strength, temperature)-The sta- 1. Preparation of Ca2’ Buffers 23 bility constants are adjusted for ionic strength, temperature, and pH as described in the earlier sections of this chapter. A. Getting Started Quickly with MAXC 1. Copy the MAXC files into a directory on your hard drive 2. Type MAXC to start the program and be prompted automatically through the following steps 3. CCM) 4. Choose a Calculation: Type “ALT C” then “Total” to calculate total [metalls from free [metalls and total [chelatorls “Free” to calculate free [metalls from total [metalls and total [chelatorls “Chelator” to calculate free and total [chelatorls from free and total [ metalls 5.
A Practical Guide to the Study of Calcium in Living Cells by Richard Nuccitelli, Leslie Wilson, Paul T. Matsudaira