Chapter title |
Ca 2+ Binding and Transport Studied with Ca 2+ /EGTA Buffers and 45 Ca 2+
|
---|---|
Chapter number | 23 |
Book title |
P-Type ATPases
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3179-8_23 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3178-1, 978-1-4939-3179-8
|
Authors |
Pankaj Sehgal, Claus Olesen, Jesper V. Møller |
Abstract |
The chapter describes procedures useful for determination of Ca(2+) binding by membranous Ca(2+)-ATPase based on the correction for the removal of Ca(2+) present in a non-bound state in the suspension medium. This is done by a filtration procedure that retains the membranous material on the Millipore filters. With suitable sucking devices it is possible to gently remove without dehydration nearly all medium from the Ca(2+) containing membranes, except that required for wetting of the filters on which they are deposited. Correction for this effect can be done with a double-filter where the radioactive content of the lower (protein-free) filter is subtracted from that present in the upper filter for calculation of Ca(2+) binding. This methodology can be used to study the effect of inhibitors on Ca(2+) binding and -transport, and with Ca(2+)/EGTA buffers to explore the Ca(2+) binding affinities and cooperative aspects of the two transport sites. |
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Unknown | 4 | 100% |
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Professor | 2 | 50% |
Researcher | 2 | 50% |
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