Osteosarcoma cybrids were permeabilized with digitonin
(4 [micro]M), and respiration was initiated with complex I substrate (malate/pyruvate, 5 mM).
A problem of using digitonin
as a solubilizing agent is that it exists as large micelles, which makes it difficult to characterize solubilized muscarinic receptors in terms of molecular mass: the ligand binding activity in a digitonin
extract was found in the fraction corresponding to a mass of around a million.
6 [micro]mol/L digitonin
for the permeabilization of the cell membranes, 1 mmol/L pyruvate, 1 mmol/L malate, and, where indicated, 2.
dodecyldimethylamine oxide, lysolecithins), and ii) detergents belonging to the group of bile salts or other steroid-based detergents such as 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfon ate (CHAPS) and digitonin
Fura-2AM, serotonin, DNA, DNaseI, guanidine hydrochloride, digitonin
and EGTA were purchased from Sigma-Aldrich Chemicals, Bangalore, India.
Kinetics of the inactivation of the muscarine cholinoreceptor, solubilized by digitonin
Cells were permeabilized in an oxygraph chamber by digitonin
addition (10 [micro]g/mL).
6]) were washed with ice-cold Dulbecco's phosphate-buffered saline and were resuspended in 100 [micro]L of ice-cold digitonin
lysis buffer (0.
Permeabilization and cell viability of PFFs treated with digitonin
and plating on trypticase soy agar (TSA).
Germinally transgenic tadpoles were produced by restriction enzyme-mediated integration nuclear transplantation according to Kroll and Amaya (1996), with the following modifications: sperm was purified by centrifugation on a two-layer discontinuous Percoll (Sigma) gradient before the permeabilization step, which was performed with digitonin
(Sigma) instead of lysolecithin.
The muscarinic acetylcholine receptors (mAChR), which are members of the family of receptors that are coupled to guanine nucleotide binding proteins (G proteins) and whose structure is characterized by the seven membrane-spanning [alpha]-helical domains [2, 3], are usually solubilized in digitonin
or in a mixture of digitonin
and sodium cholate, which have yielded more than 50% of the soluble receptors with retention of ligand binding activity [4, 5].