Important parameters in semi‐dry electrophoretic transfer

Abstract

The efficiency of semi-dry electrophoretic transfer after sodium dodecyl sulfate (SDS)-electrophoresis using PhastGel media was investigated in a model system using three isotope labelled proteins. To give a full picture of the blotting process the amount of protein present in the gel, membranes, and filter papers was determined after different transfer times. The influence of the transfer buffer, commonly used additives such as methanol and SDS, and several different immobilizing matrices was investigated. Soybean trypsin inhibitor, bovine serum albumin, and ferritin were used as model proteins to study the effect of size on transfer efficiency. Basically, all three stages of the blotting process decide the result; the elution of protein from the gel, the immobilization of protein to the membrane, and the loss of material from the membrane during transfer. A theoretical explanation for the observed poor binding to a second membrane is discussed. Our results show that the buffer composition has little influence on the efficiency of transfer from the gel, but can be significant to the binding capacity of the membrane. In all experiments performed, there was never one moment during the transfer when all protein was eluted from the gel and simultaneously still bound to the membrane. The highest recovery in the membrane was obtained at different time intervals for different proteins. This indicates that quantitative transfer procedures cannot be generalized. However, obtaining an optimal method for reliable quantification of a specific protein or group of proteins is possible. For general protein staining of nitrocellulose and polyvinylidene difluoride membranes, a highly sensitive silver staining method requiring only 15 min has been used.

Locating the barnacle settlement pheromone: spatial and ontogenetic expression of the settlement-inducing protein complex ofBalanus amphitrite

“…After rinsing with TTBS, the membranes were incubated with the secondary antibody (1 : 5000 dilution, HRPO-conjugated anti-rabbit IgG goat antibody (Chemicon)) and immunoreactive bands were detected using the Lumi-light chemiluminescence kit (Roche). The blots were then silver stained to determine the amount of protein loaded on the gel and the transfer efficiency ( Jacobson & Karsnas 1990 Expression of the SIPC protein in different developmental stages. Protein from each sample (5 mg) was immunoblotted with anti-SIPC (nZ1 experiment).…”

Isolation, Characterization, and Antimicrobial Properties of Bovine Oligosaccharide-binding Protein

“…Western Blot and Dot Blot Analyses-Acid extracts of bovine leukocytes, extracts of leukocyte granules, and purified bOBP were lyophilized, boiled for 2 min in dithiothreitol-containing SDS-PAGE sample buffer, subjected to Tricine-SDS-PAGE (12.5% acrylamide), and transferred to 0.22-m nitrocellulose membranes (MSI, Westborough, MA) using the semi-dry electroblotting technique (41). For Dot Blot analyses, RP-HPLC fractions were subjected to rotoevaporation briefly to remove acetonitrile, and they were applied in 1-l aliquots to 0.22-m nitrocellulose membranes.…”

Oxidative stress impairs function and increases redox protein modifications in human spermatozoa

“…Positive immunoreactive bands were detected using chemiluminescence (Lumi-light; Roche Molecular Biochemicals). After detection, membranes were washed with distilled water and silver stained (Jacobson & Karsnas 1990) to assure equal loading for each sample.…”