Protein Expression and Analysis

Western blots can be ugly. I mean down-right, horrifically, wall-of-shame ugly.  Not only can they be embarrassing to show to your colleagues, but the ugliness can obscure your results, making it impossible to interpret your data. Blotting consists of many experimental steps, which makes the technique naturally error-prone. Although standardized protocols exist, many fail to…

Isoelectric focusing electrophoresis (IEF) of proteins is nowhere near as popular as its cousin – sodium dodecyl sulphate-polyacrylamide gel electrophoresis aka SDS-PAGE. While in both methods the proteins are denatured, IEF is a gel-based electrophoretic separation of proteins using difference in their overall charges. The sodium dodecyl sulphate – SDS part of the usual gel…

Wouldn’t it be great to put your nucleotide sequence into a program and get back a 3D-structure of your protein and a full description of its functions? In theory, because the protein 3D-structure is determined by the aminoacid sequence, given the right algorithm and a powerful enough computer, this should be simple.  In practice, because…

Have you ever emerged from the lab, bleary-eyed, blinking dazedly at the sun after spending hours hunched over a lab bench counting endless bacterial colonies or viral plaques? A necessary evil… I consider colony/plaque counting one of the necessary evils of working with microorganisms.  Necessary because many experiments have an endpoint that requires determining the…

In the previous article on EC numbers, I explained how the Enzyme Commission names enzymes, and why it is so important. In this article I’d like to take you on a brief journey through the history of the Enzyme Commission. Like many histories in science (e.g. this!), it is fascinating and gives a useful perspective…

As a biologist you will no doubt have seen Enzyme Commission (EC) numbers. An EC number is group of four numbers separated by periods in papers discussing enzymes…something like this: EC 1.1.2.1. But do you know what these numbers mean? Or where they came from? Or why we use them? If not, I will aim…

The ELISA (enzyme-linked immunosorbent assay) is a rapid method used to detect the amount of a protein of interest in clinical and experimental samples. There are a number of ELISA formats to choose from, depending on your research needs. These include direct ELISA, indirect ELISA, competitive ELISA and sandwich ELISA. We have previously covered the…

Transfection of animal cells has proven an invaluable tool in studies of gene expression, cell behavior, cell processes and molecular genetics. Essentially, transient pores are opened within the cell’s lipid bilayer, allowing insertion of nucleic acids (DNA, RNA, siRNA, RNAi), proteins, nanoparticles, and even antibodies into the cellular milieu. Transfection can be achieved using many…

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…

LB medium is a staple in virtually every lab. It’s commonly used to propagate E. coli, and as such will be used frequently by any lab that does cloning. Chances are, LB broth or plates were one of the first things you learned to make as a newbie in the lab.  Here are a few…

Co-immunoprecipitation is a method used to detect protein-protein interactions. While it can be wonderful when it works, there are many problems associated with this technique. One of the biggest problems that I have faced when using this method is contamination by the light and heavy chains of my precipitating antibody when performing western blots of…

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,…

The last step in western blotting is imaging the blot – this is the moment of truth, when you finally get to see the results of the experiment you’ve been working on for so long!  There are a variety of different ways to image your blot.  The method you choose will largely depend on the…

The first step in most Western blotting experiments is lysing your cells to extract protein.  You need to break open your cells in order to be able to isolate the proteins, and you need to do this with the least degradation and the most reproducibility possible.  Depending on what your starting material is, there are…

You’ve masterfully run and transferred your gel, and now it’s time to probe and quantify your protein(s). You’ve got your antibodies and ECL ready to go. Substrate – check. Film cartridge – check. Darkroom – ah yes, that magical place where night vision goggles are required to navigate a veritable minefield of potential chaos. It…

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…

RNA interference (RNAi) may have originated as a defense mechanism to protect cells against foreign genes introduced by viruses. This concept has since been put to use to create a powerful experimental tool for investigating gene function in organisms. Small-interfering RNA (siRNA) libraries for investigating genome-wide function can be produced by chemical synthesis of probes…

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…

Do you want to immunoprecipitate (IP) a protein with a molecular weight that is anywhere near 55 kDa or 25 kDa? Then you have an irritating problem to deal with: antibody co-elution. But don’t panic, we have six strategies for dealing with your new problem. The Problem: Typically, the IP antibody is bound to Protein…

Sepharose beads are porous, which gives them a high surface area for interaction with proteins and allows them to hold a lot of liquid. This is perfect for the application that they were originally designed for: purifying milligrams of protein in columns. When immunoprecipitation (IP) – a small-scale technique for pulling specific proteins out of solution using…

At the end of my last article, I provided some practical tips and tricks for working with enzymes at the bench. Now, we’ll cover one of the cornerstone techniques of enzymology work: the enzyme assay. Starting with the simple assays and eventually working our way to the more complex, this article introduces the principles of…

In Part 1 of this series, we began our journey into the fascinating world of enzymology. We looked at the most basic concepts of what an enzyme is and the incredible jobs it can do. In Part 2 of “Working with Enzymes,” I will look at some things that you should keep in mind to…

In parts one and two of this series I described how semi-permeable membranes and precipitation methods could be used to concentrate your protein-of-interest, but there is one more method that you may not have thought of for protein concentration – chromatography. While chromatography resins are an obvious choice for protein purification, they can also be…