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Download the Protein Blotting Guide
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:: Posted by American Biotechnologist on 02-20-2012
We are all intimately familiar with protein blotting techniques which have been a cornerstone of the biochemicstry/biology lab for the past 30 years.
As is well known, the efficiency of protein migration is affected by various factors including the size and charge of the protein, and protocol optimization is often needed on a protein-specific basis. In fact, it can be particularly challenging to transfer large molecular weight proteins alongside small molecular weight proteins, as transfer conditions may cause small proteins to blow through the membrane.
Currently there are three popular techniques for protein transfer: the tank transfer, the semi-dry blotting method and the fast blotting “turbo” technique (for transfer within 3-10 minutes).
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.
While we are all familiar with the role of methyltransferase in DNA and protein modification in the nucleus, (think epigenetics with regards to DNA), this is the first time that methylation in the cytoplasm has been shown to promote protein complex formation.
The researchers first identified an enzyme which is mainly present in the cytoplasm and which methylates the amino acid lysine (Smyd2). Then they searched for interaction partners of the enzyme Smyd2
and found the heat shock protein Hsp90. The scientists went on to show that Smyd2 and methylated Hsp90 form a complex with the muscle protein titin.
According to the authors, “Titin is the largest protein in the human body and known primarily for its role as an elastic spring in muscle cells. Precisely this elastic region of titin is protected by the association with methylated Hsp90.”
In skeletal muscle cells of the zebrafish, the team explored what happens when the protection by the methylated heat shock protein is repressed. By genetic manipulation they altered the organism in such a way that it no longer produced the enzyme Smyd2, which blocked the methylation of Hsp90. Without methylated Hsp90, the elastic titin region was unstable and muscle function strongly impaired; the regular muscle structure was partially disrupted.
Click here for a link to the Genes and Development paper.