Download Calcium signaling protocols (Methods in Molecular Biology by David G. Lambert PDF

By David G. Lambert

David Lambert and a panel of major experts current quite a lot of experimental protocols for learning Ca2+ signaling. those optimized concepts conceal the extra universal functions, together with 45Ca2+ flux measurements, and easy fluorometric expertise, in addition to extra subtle tools, together with confocal microscopy and subcellular Ca2+ imaging. There also are equipment - principally in keeping with fluorescence dimension - to figure out Ca2+ channel task and the discharge of Ca2+ from intracellular shops. Calcium Signaling Protocols bargains modern day researchers comfortably reproducible laboratory tools that give the chance to envision the calcium signaling approach intimately in a number cells of animal and plant starting place.

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Generally longer incubations at low temperatures improve the ratio of specific-to-background labeling. 9. Remove excess antibody from cover slips by briefly washing with PBS, followed by two additional washes in PBS, each for 2 min. 60 Mongan et al. 10. Wash in PBS for 30 min at 4°C. 11. Thoroughly remove excess antibody from the cover slips using a vacuum-driven filter pump. 12. Add 60 µL of the secondary antibody to each cover slip including the control cells. Incubate for 1 hour at 37°C, overnight at room temperature, or 24 h at 4°C.

Transfer this to the centrifuge tube. 8. Sediment at 1000g in a low-speed centrifuge for 3 min. 9. Remove supernatant and resuspend the pellet into 30 mL of fresh experimental buffer. Invert the tube three times and resediment at 500g for 3 min. 10. Repeat step 9 once more, and finally resuspend the pellet in 3 mL of experimental buffer. 2. Fura-2 Loading and Measurement of Intracellular Calcium Optimal fura-2 loading time and de-esterification time may vary depending on the cell type used, and hence it is recommended that these times should be adjusted accordingly.

Biol. Chem. 258, 4876–4882. Confocal Microscopy 51 4 Confocal Microscopy Theory and Applications Laura C. Mongan, Joanne Gormally, Andrew R. D. Hubbard, Christopher d’Lacey, and Colin D. Ockleford 1. Introduction This chapter will attempt to provide an overview of the theory and details of some basic applications of the technique of confocal microscopy. Since this is intended to be a general introduction to the possibilities available, it is not appropriate to include detailed descriptions of the specialized techniques used in physiological studies leading to the imaging and measurement of Ca2+-containing samples.

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