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

Introduction

Good Western blot blocking methods are crucial in order to achieve clean and reliable results. Protein samples are first ran on a polyacrylamide gel to separate proteins by size. The proteins are then transferred to a nitrocellulose or PVDF membrane. A blocking agent is then used to prevent primary and secondary antibodies from binding to the membrane non-specifically (areas without the protein of interest). Proper blocking methods cover the membrane surface without interfering with bound protein; thus preventing non-specific antibody binding and non-specific signal (noise or background) due to the membrane’s high affinity for proteins.

Commercial stripping buffers, such as G-Bioscience’s Western ReProbe™ and Western ReProbe™ PLUS, are specifically formulated to dissociate and remove all primary and secondary antibodies from the membrane-immobilized proteins without destroying the antigenic binding sites or removing the protein. This allows for several reprobings on the same membrane which will save you time, money, and precious protein samples. Western blotting can be time consuming (protein electrophoresis (1-2 hrs) + protein transfer (0.5 hrs to overnight)), so you want to make the most of your precious membranes and proteins by stripping and reprobing your nitrocellulose or PVDF membrane. There are many advantages to stripping your membrane such as: conserving limited/expensive protein samples, using different antibodies to analyze the same sample and confirm results, compare phosphorylated and total protein on the same blot, recoup antibody for later use, run less SDS-page gels and transfers, and to remove/adjust the concentration of your antibody.