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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…
If you purify proteins expressed in E. coli, then you’re probably familiar with this scenario: you come in bright and early in the morning and inoculate your large flasks of media with the overnight culture, start shaking them at 37 °C, and now you wait. And watch. And wait some more. You can’t venture far,…
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
Diving deeper into protein kinases, explore the vast realm of known kinases, their amino acid targets, and the intricate process of target selection.
In the previous article in this series, we looked at the major players involved in protein phosphorylation: protein kinases, protein phosphatases, and target proteins. This time, we’ll glance over some of the tools that we can use to study various aspects of protein phosphorylation, focussing on a few I’ve personally come across. 1. Tools for…
The focus of my grad studies and postdoctoral research has been the analysis of proteins regulated by reversible protein phosphorylation. However, the number of unique facets in which protein phosphorylation can be studied is astounding, and is diverse as any niche of the biosciences. This article is the first in a series that will give…
If you have ever attempted to load a SDS-PAGE gel only to miss the well, stab the divider, and then watch helplessly as your sample squirts off into the wrong well, then this tip is for you. The fortunate among us are able to use pre-cast gels with the wells outlined on the gel plate,…
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.
In the early 1950’s so many new enzymes were being discovered in the burgeoning field of biochemistry that enzyme nomenclature was in danger of getting out of hand. With no guidelines on how to name enzymes, researchers simply chose their own. Some enzymes were given names, like diaphorase or Zwischenferment, that conveyed nothing about the…
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