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 [...]

If you read my previous article, you may have given IHC a shot. The chances are if you’re reading this one, it may not have worked. But don’t fret, help is at hand! This article contains a few common pitfalls and problems associated with IHC, and what you can do to try and get the [...]

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 [...]

Back in August I shared my training regimen for guesstimating the OD­600 readings of microbial cultures with superhuman accuracy. Although my method is effective, I will admit that it has two shortcomings: you need to make a separate standard curve for each container type, and guesstimation is not an officially sanctioned scientific method. But now, [...]

Immunohistochemistry (IHC) is a vital tool, not just at your wet bench but also in clinical labs the world over. IHC is used extensively in hospitals and veterinary practices in grading and studying cancers, and the results may determine what treatment a patient gets – including the controversial Herceptin! But it’s also pretty useful in [...]

Isolating RNA is one of the more finicky protocols there is, and everyone who does it has their own personal tips and tricks to successfully isolate intact RNA from their samples with consistency. Although RNA can be somewhat unpredictable since it is so labile, there are a few common problems that occur which can be logically [...]

These days, raw text data sets can be spat out at you from all manner of instrumentation. This automation is efficient, however it can give you a headache when it comes to extracting very specific information from reams of characters and numbers within a raw data file. Brute force (editing or extracting data from the [...]

Working with RNA is definitely an acquired skill.  It’s a lot more finicky than working with DNA, and requires careful attention to detail in order to avoid disastrous through RNase contamination.  Here are a few common ways to lose your hard-earned RNA:  1. Don’t keep everything on ice Keeping the temperature of all of your reagents cool is [...]

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 [...]