Solubilization and further chromatographic purification of highly purified, membrane-bound Na,K-ATPase
Biochemical and Biophysical Research Communications
|March 28, 1986
Abstract:
Highly purified membrane-bound Na,K-ATPase from pig kidney outer medulla was dissolved in the non-ionic detergent C12E8. Chromatography of the dissolved material on a DEAE matrix yielded enzymatical material having a ouabain-binding capacity of 6.9 nmoles per mg protein (measured according to Lowry et al., with bovine serum albumin as standard). This material, which after addition of lipids had the same K+-phosphatase turnover as the membrane-bound enzyme, could consist entirely of live molecules with a molecular weight of 145 kDa, a value close to that expected for alpha beta-promoters of Na,K-ATPase.
More Related Videos
Related Concept Videos
Detergent Purification of Membrane Proteins
Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and...
Dialysis
Dialysis is a diffusion-based purification process that separates analyte molecules from a complex matrix. This is accomplished by allowing molecules in the solution to pass through a semipermeable membrane into a liquid on the other side. The membrane is usually made of cellulose acetate or cellulose nitrate, and the second liquid must be miscible with the solution. Ions (e.g., chloride or sodium) or organic molecules (e.g., glucose) can pass through the membrane pores, which generally have...
Downstream Processing
Downstream processing begins once fermentation is complete and involves a series of steps to recover and purify products such as acids, vitamins, antibiotics, or proteins.Cell HarvestingFor example, for intracellular protein-based products, the first step is harvesting the cells. This is typically achieved using centrifugation or filtration to separate the cells from the liquid phase.Cell Disruption for Intracellular ProductsIf the target product is intracellular, the harvested cells must be...


