Are you getting the most from your western blot data? Does Imaging technique matter? Film or Imager? In this video, we describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods. For the best resolution, watch the video in full screen at HD resolution.
Posts Tagged ‘western blotting’
Bio-Rad’s new Clarity western ECL substrate provides high sensitivity (see Figure below), long signal duration, and low background even at long exposures. This combination of bright, long signal with low background makes it the perfect choice for routine use on film and digital imagers, even when re-imaging and multiple exposures are required.
The Clarity western ECL substrate is compatible with any HRP-conjugated secondary antibody and is offered in 200 and 500 ml size kits.
Western blotting, the backbone of protein research, is a universal lab procedure. While the premise of a western blot is simple — target proteins are identified and quantitated via antibody-antigen interactions — the traditional workflow is labor intensive and time consuming. Researchers have long sought a faster solution — an archetype that would streamline the entire process of separation, transfer, and visualization of results without compromising data quality. Has anybody been successful at this endeavor? Yes, a chosen few have! Want to become part of this exclusive club?
Find out more about a revolutionary new workflow that will help you cut your western blotting time in half!
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.
- Blocking was incomplete
- Increase the concentration of blocker
- Increase the duration of the blocking step
- Use a different blocking agent
- Blocker was impure
- Use a pure protein such as BSA or casein as a blocker
- Wash protocols were insufficient
- Increase the number, duration, or stringency of the washes
- Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween 20
- Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding
- The blot was left in the enzyme substrate too long (colorimetric detection)
- Remove the blot from the substrate solution when the signal-to-noise level is acceptable, and immerse in diH2O
- Contamination occurred during electrophoresis or transfer
- Discard and prepare fresh gels and transfer solutions
- Replace or thoroughly clean contaminated foam pads if a tank blotter was used
- Excessive amounts of protein were loadedon the gel or too much SDS was used inthe transfer buffer. Proteins can pass through the membrane without binding and recirculate through a tank blotting system.
- Reduce the amount of protein on the gel or SDS in the transfer buffer
- Add a second sheet of membrane to bind excess protein
- The primary or secondary antibody was too concentrated
- Increase antibody dilutions
- Perform a dot-blot experiment to optimize working antibody concentration
- Incubation trays were contaminated
- Clean the trays or use disposable trays