Keeping it constant: A lesson in protein transfer

Power supplies that are used for electrophoresis hold one parameter constant (either voltage, current, or power). The PowerPac™ HC and PowerPac Universal power supplies also have an automatic crossover capability that allows the power supply to switch over to a variable parameter if a set output limit is reached. This helps prevent damage to the transfer cell.

During transfer, if the resistance in the system decreases as a result of Joule heating, the consequences are different and depend on which parameter is held constant.

Transfers Under Constant Voltage
If the voltage is held constant throughout a transfer, the current in most transfer systems increases as the resistance drops due to heating (the exception is most semi-dry systems, where current actually drops as a result of buffer depletion). Therefore, the overall power increases during transfer, and more heating occurs. Despite the increased risk of heating, a constant voltage ensures that field strength remains constant, providing the most efficient transfer possible for tank blotting methods. Use of the cooling elements available with the various tank blotting systems should prevent problems with heating.

Transfers Under Constant Current
If the current is held constant during a run, a decrease in resistance results in a decrease in voltage and power over time. Though heating is minimized, proteins are transferred more slowly due to decreased field strength.

Transfers Under Constant Power
If the power is held constant during a transfer, changes in resistance result in increases in current, but to a lesser degree than when voltage is held constant. Constant power is an alternative to constant current for regulating heat production during transfer.

The above information was adapted from Bio-Rad’s protein blotting guide. For more great information, be sure to download the Protein Blotting Guide from Bio-Rad Laboratories.

Protein Standard Selection Guide

In a previous post we provided you with a guide for choosing the ideal protein standard tailored to your specific experimental application. Whether you are looking for molecular weight (MW) determination, standards that light up during your western blot chemiluminescent development or antibodies that fluoresce, you will find some interesting pointers contained in that post.

If you prefer all-in-one posters and visually stimulating charts, you should definitely check out Bio-Rad’s latest protein standards selection guide. The guide presents ten different protein-based experiments and has a comprehensive chart for helping you figure out which protein standard to use based on the size of your protein of interest and the application you are using it with. Click on the picture below to download the most recent version of the guide.

How to Choose the Ideal Protein Standard

In the following post, we will share with you a practical guide for selecting appropriate protein standards. The information was distilled from an article published in issue 127 of Bio-Rad Laboratories’ BioRadiations Magazine and can be found in its entirety on page 11 when clicking on this link. The guide will be broken down into both gel electrophoresis and western blotting applications.

Gel Electrophoresis
Unstained Standards
When using gel electrophoresis for molecular weight (MW) determination, unstained standards are the ladders of choice. Unstained standards are unencumbered by protein dyes and therefore give the most accurate electrophoretic mobility pattern. They are also required when a fluorescent dye is used to stain the gel or blot to visualize total protein.

Movie star of the week: V3 Western Workflow™

Bio-Rad Laboratories V3 Western Workflow™ makes the BioTechniques video of the week. Check it out by clicking on the picture below (video will open in a new window).

Bio-Rad Cuts Western Blotting Time in Half with New V3 Western Workflow

Bio-Rad Laboratories, Inc. launched its V3 Western Workflow, a portfolio of products designed to reassure researchers at every step throughout the immunoblotting workflow. Leveraging stain-free imaging technology, the V3 Western Workflow enables scientists to visualize protein separation, verify protein transfer, and confidently validate blot data via total protein normalization.