Protein Expression and Analysis

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…

While precipitation is an obvious choice for concentrating DNA and RNA samples, it can also be an effective way to concentrate proteins. Here in installment two of this three part series, I describe the two most common methods for protein precipitation – ammonium sulfate and trichloroacetic acid. Background Precipitation of proteins occurs primarily by hydrophobic…

This is the first of a three part series describing some of the most common methods for concentrating proteins. In later installments I’ll discuss using protein precipitation and chromatography to concentrate a protein. However, here I’ll detail the most popular approach – semi-permeable membranes, used for both dialysis and commercial protein concentrators. Structure of the…

Protein kinases and protein phosphatases phosphorylate and dephosphorylate a plethora of proteins. They are responsible for regulating the majority of cellular activities. Because of their importance, they can seem intimidating to tackle as a research project. At the end of the day however, kinases and phosphatases are- simply put- enzymes. Therefore, you can standard enzyme…

Chris Dieni wraps up his series on the Basics of Protein Phosphorylation with this final part on further reading and resources available.

Chris Dieni explores the promiscuous yet intriguing nature of protein phosphatases

In my last article, I introduced Cell-Free Protein Synthesis. Today I want to talk about a major bottleneck in in vitro cell-free protein expression; low yield. Most often, paying attention to the important factors such as template purity and design, transcription and translation inhibitors, and potassium and magnesium concentration will solve any problems with low…

Cell-free protein synthesis (aka In vitro translation) refers to protein production in vitro using lysates generated that provide the cellular machinery necessary for synthesis. The lysates can be of bacterial or eukaryotic origin. It is a useful alternative to in vivo synthesis for generating protein for the study of things like: So what are the…

You are not alone. Everyone makes a hash of their protein gel sometimes but this resource can help you work out what went wrong, and feel better for seeing gels even worse than yours.

This is the story of how one of the most famous and quirky naming conventions in biology came into being.

An introduction to the Monash protein folding database, which contains protocols for refolding over 600 different proteins from inclusion bodies.