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

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.

There are a lot of reasons why researchers all over the world choose enhanced chemiluminescence (ECL) detection for a wide range of Western blot applications. ECL is considered as the experts’ detection method of choice due to its high sensitivity, excellent signal-to-noise ratio, and wide dynamic range. Additionally, ECL is also useful in quantifying a wide variety of biological materials such as cell RNA, DNA and other analytes. 

What is protein enrichment and why is there a need for it? Basically, protein enrichment is a technique where proteins of interest in a biological sample are concentrated to make them more suitable for identification and downstream analysis.

Detergents form micelles which can trap hydrophobic molecules into these micelles and allow the extraction of membrane proteins through solubilization.  The “Critical Micelle Concentration” or CMC of a detergent is the concentration of a detergent in which micelles start to form.  Detergents belong to a class of compounds called surfactants.  They are indispensable when working with integral membrane proteins and are able to partition into biological membranes, extract proteins, and maintain protein solubility in solution.  Detergents are useful in a wide variety of applications as well including PAGE, inclusion body solubilization, and lipid raft preparation.  Determining the CMC allows you to choose which detergent may be best for a particular application.  This can be done by a variety of methods including light scattering or measuring the surface tension, both of which can be time consuming and expensive.  A simple method is by plotting optical density of solubilized dye against detergent concentration described by Brigitte Vulliez‐Le Normand and Jean‐Luc Eisele.  G-Biosciences' Optimizer blueBALLS™ uses a similar principle to help determine CMC.