Correction For Nonspecific Binding Research Articles

Hydroxylapatite “batch” assay for estrogen receptors: Increased sensitivity over present receptor assays

A “batch” hydroxylapatite procedure for the adsorption of the uterine estradiol 17β-receptor complex is described. Characterization with respect to washing efficiency, binding specificity, competition, adsorption time, sensitivity and stability against increasing KC1 ionic strength were included. Equilibrium parameters obtained by Scatchard analysis were compared to the range of values found in the literature. K ta and receptor site concentration per uterus obtained by this “batch” technique were found to be well within the range described by these reported values. This technique is particularly advantageous due to its wide range of operational sensitivity (capable of detecting specific estradiol-17β binding to a cytosol fraction containing from 5 to 500 μg protein per 225 μl). The assay is run entirely at low temperature (0–2°C). In addition this technique depends on a homogeneous insoluble chemical, hydroxylapatite, which can be obtained in analytical grade quantities of uniform particle size, shows little affinity for free steroid, can be readily packed or resuspended, and appears independent of changes in concentrations of KC1 up to 2500 mM. Additional considerations include the effect of temperature during assay, the importance of empirical correction for non-specific binding, the contributions of binding information on the calculation of equilibrium parameters and statistical evaluation of random error and assay repeatability.

Biochemical studies of tast sensation. Binding of L-[3H]alanine to a sedimentable fraction from catfish barbel epithelium.

Large numbers of taste buds are distributed over the body surface of the channel catfish ictalurus punctatus, with the barbels having an especially high density. L-Alanine, as well as certain other amino acids, are taste stimuli in this animal. Epithelial tissue obtained by gentle scraping of the barbel surface was fractionated by differential centrifugation. A sedimentable fraction (P2) was prepared that was enriched in L[OH]alanine binding activity, the plasma membrane marker enzyme 5'-nucleotidase, and the mitochondrial marker succinate cytochrome c reductase, but not the microsomal marker NADH cytochrome c redu.ctase. Binding of L-[OH]alanine was measured using a Millipore filter method in which correction for non-specific binding was also determined. Time, temperature, and pH for measuring binding activity were established. At the optimal pH of 7.8, the KD for L-alanine is 4.8 X 10(-6) M. The first order dissociation rate constant at 6 degrees is 3.8 X 10(-4) s-1 and at 24 degrees it is 12.1 X 10(-4) s-1. The second order rate constant for association is between 10(2) and 10(3) M-1 S-1. Reversibility of the binding interaction was also demonstrates by the rapid displacement of bound L-[3H]alanine by a large excess of unlabeled L-alanine. That the binding does not represent incorporation into protein was confirmed by the lack of effect of puromycin. The amounts bound of several other chemostimulatory amino acids werealso determined.