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Archive for the ‘Bio-Rad Tutorial’ Category

Keeping it constant: A lesson in protein transfer

 :: Posted by American Biotechnologist on 05-22-2012

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.

Running a great 2D gel from start to finish

 :: Posted by American Biotechnologist on 04-02-2012

Great proteomics tutorial from Bio-Rad Laboratories.

Introduction to High Resolution Melt Analysis

 :: Posted by American Biotechnologist on 03-05-2012

In our last post we told you about how Bio-Rad Laboratories very own Sean Taylor and Francisco Bizouarn were crowned the kings of MIQE. Today we’d like to bring you another classic from his majesty Frank. In the slideshow below, you will learn the basics of High Resolution Melt Analysis (HRM), applications, important considerations, assay design and optimization and analysis software. Enjoy. And all hail the king!

A primer on fluorescence detection

 :: Posted by American Biotechnologist on 01-31-2012

Yesterday we told you about how to get more data from your western blots by utilizing multiplex fluorescent detection. Today, we will provide you with a primer on fluorescent detection taken from the Bio-Rad Laboratories Protein Blotting Guide.

In fluorescence, a high-energy photon (ℎVex) excites a fluorophore, causing it to leave the ground state (S0) and enter a higher energy state (S’1). Some of this energy dissipates, allowing the fluorophore to enter a relaxed excited state (S1). A photon of light is emitted (ℎVem), returning the fluorophore to the ground state. The emitted photon is of a lower energy
(longer wavelength) due to the dissipation of energy while in the excited state.

When using fluorescence detection, consider the following optical characteristics of the fluorophores to optimize the signal:

  • Quantum yield — efficiency of photon emission after absorption of a photon. Processes that return the fluorophore to the ground state but do not result in the emission of a fluorescence photon lower the quantum yield.Fluorop hores with higher quantum yields are generally brighter
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  • Extinction coefficient — measure of how well a fluorophore absorbs light at a specific wavelength. Since absorbance depends on path length and concentration (Beer’s Law), the extinction coefficient is usually expressed in cm–1 M–1. As with quantum yield, fluorophores with higher extinction coefficients are usually brighter
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  • Stokes shift — difference in the maximum excitation and emission wavelengths of a fluorophore. Since some energy is dissipated while the fluorophore is in the excited state, emitted photons are of lower energy (longer wavelength) than the light used for excitation. Larger Stokes shifts minimize overlap between the excitation and emission wavelengths, increasing the detected signal
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  • Excitation and emission spectra — excitation spectra are plots of the fluorescence intensity of a fluorophore over the range of excitation wavelengths; emission spectra show the emission wavelengths of the fluorescing molecule. Choose fluorophores that can be excited by the light source in the imager and that have emission spectra that can be captured by the instrument. When performing multiplex western blots, choose fluorophores with minimally overlapping spectra to avoid channel crosstalk
  • For more information be sure to download the Protein Blotting Guide from Bio-Rad Laboratories.

Increase Western Blot Throughput with Multiplex Fluorescent Detection

 :: Posted by American Biotechnologist on 01-30-2012

The most common method for analyzing protein expression levels is western blotting with detetion of a single protein target, using horseradish peroxidase-conjugated or alkaline phosphatase-conjugated antibody probes combined with colorimetric or chemiluminescent detection. While these methods work well for studying a single target, they are unsuitable for anlayzing multiple targets at the same time, particularly if the target proteins are of unknown or similar sizes. For analysis of multiple targets, the blot is typically stripped and reprobed for additional targets of interest. Reprobing is time consuming, and often some of the target protein on the blot is lost as a result of the stripping procedure. If one protein is removed to a greater of lesser extent relative to another protein, the ability to quantitate the relative amounts of diffferent proteins of interest is compromised.

In this technical note, you will be introduced to fluorescent western blotting detection which is superior to traditional western blotting when trying to analyze multiple proteins.

Advantages include:

  • fast and quantitative detection of multiple proteins in a single experiment
  • sensitivity compared to chemiluminescent detection
  • linear dynamic range up to 10 times greater than that of chemiluminescent detection
  • fewer experimental steps than chemiluminescent detection
  • no substrate requirement, and therefore no risk of exhausting the substrate and causing a “dead zone” in the blot
  • the ability to visualize and quantitate both phosphorylated and non-phosphorylated forms of individual proteins

The technical note is divided into three sections to help those who are new to fluorescent western blot detection quickly generate reliable and reproducible results.

Click here to download the the technote now!