Bio-Rad has sponsored the development of
this site to advance the productivity of the American Biotechnology sector and the fine people who
work in it across the country. We invite readers to contribute content:
posters, tools, research and presentations, articles white papers, multimedia, music
downloads and entertainment, conference announcements, videos. Please contact email@example.com more information.
Download the Protein Blotting Guide
Download the Stem Cell Guide for Life Science Researchers
:: 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.
:: Posted by American Biotechnologist on 01-19-2012
So you’ve isolated your protein, ran them on a gel and now you’re ready to transfer them to a membrane to begin western blotting. Sounds simple, right? Not so fast. Don’t forget to equilibrate your gel prior to beginning your transfer. Gel equilibration generally involves rinsing the gel in diH2O and soaking it in transfer buffer for approximately 15 min. While it may sound simple, (and it truly is), it is a step that might make the difference between an ugly blot and one that is publication worthy.
Below are some points to consider about gel equilibration:
Gel equilibration removes contaminating electrophoresis buffer salts. If not removed, these salts increase the conductivity of the transfer buffer and the amount of heat generated during transfer.
Equilibration also allows the gel to adjust to its final size prior to electrophoretic transfer. Gels shrink or swell to various degrees in the transfer buffer depending on the acrylamide percentage and the buffer composition.
Equilibration is not necessary (i) when the same buffer is used for both electrophoresis and transfer (for example, native gel transfers), or (ii) when using rapid semi-dry transfer systems such as the Trans-Blot® Turbo™ system (consult the user manual for the system you are using).
:: Posted by American Biotechnologist on 01-12-2012
So you think that protein transfer for western blotting is a piece of cake? Consider these important tips before proceeding:
Use only high-quality, analytical grade methanol. Impure methanol can increase transfer buffer conductivity and yield a poor transfer.
In many cases, ethanol can be substituted for methanol in the transfer buffer with minimal impact on transfer efficiency. Check this using your samples.
Do not reuse transfer buffer since the buffer will likely lose its ability to maintain a stable pH during transfer.
Do not dilute transfer buffers below their recommended levels since this decreases their buffering capacity.
Do not adjust the pH of transfer buffers unless specifically indicated. Adjusting the pH of transfer buffers can result in increased buffer conductivity, manifested by higher initial current output and decreased resistance.
Increasing SDS in the transfer buffer increases protein transfer from the gel but decreases binding of the protein to nitrocellulose membrane. PVDF membrane can be substituted for nitrocellulose when SDS is used in the transfer buffer.
Addition of SDS increases the relative current, power, and heating during transfer, and may also affect antigenicity of some proteins.
Increasing methanol in the transfer buffer decreases protein transfer from the gel and increases binding of the protein to nitrocellulose membrane.