A silver-staining procedure for enhancing the sensitivity of protein detection on nitrocellulose membranes in immunoblotting is described. After completing any peroxidase-Ni-diaminobenzidine immunostaining, nitrocellulose sheets are placed in a physical developer, containing sodium tungstate and ascorbic acid, until the desired grade of silver-intensification has been reached. In this way a 16- to 64-fold amplification of intensity of the initial immunostaining can be achieved. False positive silver staining of protein bands and of background are suppressed by replacing bovine serum albumin, the blocking agent most frequently used in immunoblotting, with skimmed milk.

3,3'-Diaminobenzidine↗

PubMedDate not supplied

Ion and water distribution in pig lenses incubated at 0 degree C to disable ion transport pumps.

This study was designed to test how extended exposure of lenses to sera with different ionic strengths influences the distribution of ions and water in the lens. Pig lenses were incubated in cold sera (0 degree C), which were adjusted to variable concentrations of NaCl, and their K+, Na+, Cl-, and water contents were measured. Incubation at 0 degree C inhibits active transport processes and thereby allows equilibration of the mobile ions and water. The hypothesis was that lens water content (volume) would follow the ion-induced protein changes predicted by a model derived from previous osmotic studies on proteins. As expected, exposure of the lens to cold caused a gain of sodium and a partial loss of potassium. However, the potassium concentration in the lens remained several fold higher than that in the bathing solution (about 41 vs. 1.8-4.6 mM/kg H2O), indicating that a portion of the potassium within the cold-exposed lens was not free to diffuse. That the water content of the lens showed a negative rather than a positive relationship with the concentration of NaCl within the lens was explained by the idea that an increase in NaCl within the lens (up to at least 250 mM/kg H2O) causes a decrease in the osmotically unresponsive water volume associated with lens proteins.

Animals↗