Posts tagged ‘Western blot’
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.
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.
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.
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.
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).