The ELISA assay is a rapid method used to detect the amount of a protein of interest in experimental samples. While this process has become increasing automated and less labour intensive, it is still important to carry out these assays with care to maintain accuracy and maximise the usability of your results. The following are [...]

In her article How to Get Perfect Protein Transfer in Western Blotting, Emily Crow recommends Coomassie staining your gel after transfer to the membrane to check the quality of the transfer. A good transfer should not leave behind proteins and PVDF membrane, stained by 0.1% Ponceau S in  5% phosphoric acid and destained with water [...]

Finding a good commercial primary antibody can often feel like playing Russian roulette.  Nothing is more disappointing than buying a $300 antibody that doesn’t work for your use.  There are some steps you can take, however, to increase your likelihood of success. 1)   Scout out other labs.  Before you buy, ask if anyone around you [...]

Tandem affinity purification (TAP) is a versatile technique allowing the isolation of proteins for various purposes including Western blot and mass-spectrometry. The target protein is fused with protein A from streptococcus and the calmodulin binding domain, which together comprise the TAP-tag (for an introduction to TAP-tagging, see this article). To purify a TAP-tagged protein from [...]

Staining proteins following SDS-PAGE or 2-dimensional electrophoresis is a very useful technique for visualising a global population of proteins or checking expression of your recombinant proteins, but how do you know which stain to use? This article explains the pros and cons of two of the most common protein staining techniques, silver staining and coomassie [...]

Protein tags are invaluable tools for the modern biologist, particularly if you work on one of the 99% of proteins for which there isn’t a nice antibody readily available. If you want to purify large amounts of your protein of interest, detect it by western blot or fluorescence microscopy, or identify its potential binding partners, [...]

Proteases: wild, mysterious, destructive.  What are these untamed elements ravaging your precious lysate? How can a drop of EDTA or a smidge of “cocktail” protect that sample, which is gently cradling your hopes, your dreams, and your desire to survive the next lab meeting? Brace yourself for a biochem flashback: in this article, we’ll explain [...]

Western blots may be great for visualizing protein expression, but they can be a perfect way to waste your precious antibody stocks if you follow the normal protocol. Thankfully, you don’t have to follow the normal protocol any more; here’s how to get great blots with a fraction of the antibody usage. I have tried [...]

When running a quantitative Western blot, it’s crucial that your sample preparation is consistent.  Incomplete protein extraction from one sample will skew your results when you compare it to the protein content of a sample that was extracted more thoroughly.  And after the protein extraction, it’s important to handle the samples in an identical manner [...]

When it comes to choosing a molecular weight marker to run on your SDS-PGE gels, there are a lot of options out there.  How do you know which one is right for you?  Read on for tips on what to consider when choosing a standard for your protein gels. Before you go about selecting a [...]