Single Cell Western Blot

The Answer to Fading Western Blots

Publications have become very strict when it comes to publishing research that includes images of blots, often requiring images of the full blot, uncut. To keep the membranes uncut, researchers often reprobe which can cause the marker to fade.

Bio-Rad has launched the brightest protein marker available to researchers which addresses such challenges. The Precision Plus Dual Protein Standard is not only brighter, but also stays on the membrane longer. This allows researchers to identify proteins more effectively and have greater confidence in their western blots.

The strong marker persistence of the Precision Plus Protein™ Dual Color Standards throughout electrophoresis and western blotting, even during rigorous stripping and reprobing protocols, makes the product a more effective tool for monitoring separation and estimating protein molecular weights.

“Providing the images of the entire blot is good practice for science and publications,” said Dr. Federica del Monte, assistant professor of medicine at Harvard Medical School. “In order to reprobe for the loading control and keep the membranes uncut, we sometimes need to strip them. This often results in fading of the markers. The ability to publish clear blots with the marker still on is very important.”

Key benefits of the new Precision Plus Protein Dual Color Standards include:

• Increased brightness — better identification of target proteins on gels and blots as well as easier monitoring of gel electrophoresis and confirmation of transfer quality

• Stronger marker persistence — marker remains on the blot even under the most rigorous conditions, boosting confidence in your western blots; stronger marker persistence also facilitates accurate molecular weight estimation

Bands in the Precision Plus Protein Dual Color Standards range from 10 to 250 kD with sharp pink reference bands at 25 and 75 kD for simple visualization. The new Dual Color Standards share the same migration pattern as their previous iteration.

Western blot basics

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.