The Protein Man's Blog | A Discussion of Protein Research

A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate. The blocking buffer is effective if it improves the sensitivity of an assay by reducing background interference and improving the signal-to-noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding.

Introduction

While agarose beads have been traditionally used for immunoprecipitation (IP) and other micro-scale protein and antibody purification procedures, magnetic beads have since taken lead and are now considered tool for protein immunoprecipitation.

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a common laboratory technique in which proteins are separated by their size by running the proteins through a polyacrylamide matrix by applying an electrical field across the matrix. A native gel electrophoresis is when there aren’t any detergents present while running the assay. This means that the factors affecting the rate of migration for proteins in a native gel electrophoresis assay are the protein’s molecular radius and total net charge across the protein. The molecular radius is determined by the complex tertiary structure and the net charge is the sum of all the positive and negative charges across all the protein’s amino acids. While native gel electrophoresis has its uses, it can be very challenging to interpret, proteins can migrate to either of the electrodes and can separate variably based on their tertiary shape. For these reasons most electrophoresis assays are run with some detergent present (usually SDS) so that only one factor (protein size) influences the rate of migration.