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 [...]

So, you’ve done your experiment, prepped your samples, and run your SDS-PAGE gel.  Now it’s time for the all-important transfer step, that tricky point that will determine the quality of your Western blot. Transfer times are empirical and based on your own particular samples, which means that there is no easy way to determine how [...]

I think that transferring Western blots is one the most enjoyable tasks to do in a lab: it’s quick, it’s messy, and on some gleeful level, it feels like a child’s art project gone wrong.  Of course, it’s also finicky and slippery and prone to tiny pitfalls that can noticeably affect the quality of your [...]

For Western blot data to be reliable, it is important that you load known amounts of sample into each lane of the gel.  This is of particular importance if you are doing a quantitative blot, where you really need to be able to compare band intensity in each sample.  In this article, we’ll talk about [...]

Tandem affinity purification is a development of existing techniques for purifying protein complexes from cells in physiological conditions. It was first described over ten years ago and has become a commonplace laboratory tool. In this brief article I’ll introduce the basic technique and describe some of its advantages. Biology is a team game. Most biological [...]

ELISAs are often used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones in research and diagnostics. While it has become easier to perform ELISAs, thanks to the various commercially available kits, there are still a number of common mistakes that can ruin your experiment.  Here are my top 5 top simple [...]

When purifying a protein, it’s important to keep your protein happy; that usually means that the protein is soluble and active.  Creating a buffer that prevents unfolding and aggregation is therefore crucial to the success of your experiments.  There are several factors that you should consider when designing a buffer: pH, buffering system, salt, reducing [...]

Have you ever wished you could transfer the same SDS-PAGE gel twice? Sometimes, when you are blotting for many different proteins of similar size, stripping and reprobing multiple times can become impractical.  Here’s a simple diffusion transfer method that can be used to generate duplicate membranes from a single gel: Take a glass plate, or [...]

Phosphorylation is one of the major post-translational modifications that regulate the activity of a protein. Around a third of human proteins are believed to be phosphorylated, and so the kinases and phosphatases that mediate protein phosphorylation are of major interest to biomedical researchers. However detecting protein phosphorylation can be difficult, particularly from cell extracts. Phospho-specific [...]

Accurate transfer of proteins from the SDS-PAGE gel to your membrane is an important step in Western blotting.  However, optimizing transfer times is hit-or-miss, and it can take several tries to get a publication-worthy image.  Here are a few hints on how to ensure that your transfer is accurate and complete: Always include a pre-stained [...]