By Anthony R. Means, P. Michael Conn (Eds.)
The significantly acclaimed laboratory normal, Methods in Enzymology, is without doubt one of the such a lot hugely revered courses within the box of biochemistry. because 1955, every one quantity has been eagerly awaited, often consulted, and praised by means of researchers and reviewers alike. The sequence includes a lot fabric nonetheless suitable at the present time - really a necessary ebook for researchers in all fields of existence sciences
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Additional info for Cellular Regulators Part A: Calcium- and Calmodulin-Binding Proteins
Gel-Permeation Chromatography The calregulin peak obtained from the Con A-agarose/HPLC-ion exchange column is concentrated with Amicon P-10 membrane and loaded on HPLC TSK-G3000SWG column. 1), containing 150 mM NaCI and 1 mM DTT. Calregulin is eluted as a major peak of calcium-binding activity as analyzed by Chelex assay and is free from other contaminants. Results from a representative purification scheme are summarized in Table I. From 1 kg of bovine liver, 72 mg of calregulin is obtained. 3%.
Coating of Anti-Calregulin Antibodies on Pansorbin Cells To the 10% (w/v) suspension of pansorbin cells (Staphylococcus aureus) (Calbiochem), 1 vol (10 ml) of purified IgG fraction of anti-calregulin antiserum is added and thoroughly mixed on an end-to-end shaker for 1 hr at room temperature. The suspension is centrifuged at 1000 g for 10 min and the pellet of pansorbin cells is washed three times with 5 vol of assay buffer. The washed pellet is resuspended in the assay buffer at a final concentration of 1% w/v.
5 x 30 cm column of Affi-Gel Blue (Bio-Rad), previously equilibrated with the above 42 ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS  buffer. The IgG fraction is collected by eluting with the same buffer and collecting the flow-through fraction. Analysis of the purified fraction by 10% S D S - P A G E revealed only heavy and light chains characteristic of IgG. Radioiodination of Calregulin Purified calregulin is radioiodinated by the method of Markwell. 5, and are dried on Whatman #52 paper.
Cellular Regulators Part A: Calcium- and Calmodulin-Binding Proteins by Anthony R. Means, P. Michael Conn (Eds.)