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Posts Tagged ‘Protocols’
How to avoid leaky SDS-PAGE gels
:: Posted by American Biotechnologist on 06-03-2013
A SMART(er) way to track influenza
:: Posted by American Biotechnologist on 07-11-2012Brown University researchers have created a reliable and fast flu-detection test that can be carried in a first-aid kit. The novel prototype device isolates influenza RNA using a combination of magnetics and microfluidics, then amplifies and detects probes bound to the RNA. The technology could lead to real-time tracking of influenza. Results are published in the Journal of Molecular Diagnostics.
Spliceman to the rescue
:: Posted by American Biotechnologist on 03-12-2012In a brief paper in the journal Bioinformatics, Brown University researchers describe a new, freely available Web-based program called Spliceman for predicting whether genetic mutations are likely to disrupt the splicing of messenger RNA, potentially leading to disease.
“Spliceman takes a set of DNA sequences with point mutations and computes how likely these single nucleotide variants alter splicing phenotypes,” write co-authors Kian Huat Lim, a graduate student, and William Fairbrother, assistant professor of biology, in an “application note” published in advance online Feb. 10. It will appear in print in April.
Spliceman can be found at fairbrother.biomed.brown.edu/spliceman.
Introduction to High Resolution Melt Analysis
:: Posted by American Biotechnologist on 03-05-2012In 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 01-31-2012Yesterday 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
- 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
For more information be sure to download the Protein Blotting Guide from Bio-Rad Laboratories.