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Download the Protein Blotting Guide
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:: Posted by American Biotechnologist on 03-06-2012
Proteomics is about to take a big leap forward, that is if the NIH can help it.
Last week, the NIH put out a request for information aimed at determining how best to accelerate research in disruptive proteomics technologies. The organization is hoping that submissions will aim to greatly outperform current mass spec technologies and introduce an all new way of advancing proteomic questions.
According to the proposal:
The Disruptive Proteomics Technologies (DPT) Working Group of the NIH Common Fund wishes to identify gaps and opportunities in current technologies and methodologies related to proteome-wide measurements. For the purposes of this RFI, “disruptive” is defined as very rapid, very significant gains, similar to the “disruptive” technology development that occurred in DNA sequencing technology.
These are exciting times for the field of proteomics. Don’t be left behind! Click here to find out more on how to get involved today!
Several neurodegenerative diseases – including Alzheimer’s and ALS (Lou Gehrig’s disease) – are caused when the body’s own proteins fold incorrectly, recruit and convert healthy proteins to the misfolded form, and aggregate in large clumps that gum up the works of the nervous system. Now scientists have developed an algorithm that can predict which regions of a protein are prone to exposure upon misfolding, and how mutations in the protein and changes in the cellular environment might affect the stability of these vulnerable regions. These predictions help scientists gain a better understanding of protein dynamics, and may one day help in developing treatments to effectively combat currently incurable neurodegenerative diseases.
The algorithm uses the energy equations of thermodynamics to calculate the likelihood that certain stretches of protein will be displayed when the protein misfolds. Since the exposed regions are specific to the misfolded version of the protein, researchers can use these regions as targets for diagnostic and therapeutic treatments. The algorithm can be adapted for different proteins and predicts several potential target regions for each protein. The group has used it to study neurodegenerative disease-causing proteins as well as misfolded proteins that have been implicated in some cancers.
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
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
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
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
:: 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.
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.
:: Posted by American Biotechnologist on 01-25-2012
I’m always on the lookout for new ways of teaching proteomics. Here’s a gem that I found on YouTube.
Directed in 1971 by Robert Alan Weiss for the Department of Chemistry of Stanford University and imprinted with the “free love” aura of the period, this short film continues to be shown in biology class today. It has since spawn a series of similar funny attempts at vulgarizing protein synthesis. Narrated by Paul Berg, 1980 Nobel prize for Chemistry.