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If you’ve read our article, An Overview of Yeast Two-Hybrid (Y2H) Screening, you’ll know that one major limitation of conventional Y2H is that your protein-protein interaction must occur in the nucleus for the reporter gene to be activated. So what do you do if your protein is a receptor tyrosine kinase? Or a G protein–coupled…
Most eukaryotic proteins exist as several isoforms, differing in posttranslational modifications, which allows them to perform slightly different functions or the same function under slightly different conditions. A common posttranslational modification of proteins is glycosylation.
Agroinfiltration is a method for the transient expression of your protein of interest in a plant system. You can use it for the production of recombinant proteins or simply to determine the sub-cellular localization of your protein.
We’re already gone through the basics of how gel electrophoresis work, compared common gel types like agarose and polyacrylamide and even explored some alternatives. Now let’s look at the native versus denaturing gels. You’ll be a speGEList in no time! Denaturing Gels We’ll start with this one, as it’s very self-explanatory. Denaturing gels are exactly…
In the previous installment of this series on western blotting, we addressed potential sources of error when your final product is completely bare. But alternatively, what do you do when too much background is the problem? You may have beautiful bands of interest—but if there is a bunch of non-specific binding, your quantification and data…
Western Blotting is a long established method for which the protocol varies little from lab to lab. However, there are kits available and some tweaks that can be made to the protocols that may improve your results and reduce the time it takes you to execute this popular technique. Save Time by Co-Incubating the Primary…
As a protein biochemist and a Ph.D. student, I was given the task to express a eukaryotic protein in a bacterial system. And to say that I was having a hard time would be an understatement. It took me many PCRs, cloning and transformations to get to the right construct that would eventually express the desired…
Monoclonal antibodies: You’ve probably heard a lot about them. Unsurprisingly, you may have also used them in your research. These antibodies (mAbs) are classically produced by the hybridoma technology pioneered by Köhler and Milstein in 19751: A mouse is immunized with the substance against which you need to produce an antibody. The mouse spleen cells (consisting…
The ELISA (enzyme-linked immunosorbent assay) is arguably one of the most important and versatile tools in the toolbox of molecular biologists, biochemists and diagnosticians across the world. Defined by its simplicity and speed, the assay is easy to learn and perform in as few as five steps. But with so few variables to manipulate, an…
Let’s say you work with a gene, and it has wonderful potential. Excitedly, you throw your gene into the cells and voila! It’s there. Great. Now what? Genes are powerful tools for directing cell activity, but thanks to that curiosity characteristic of scientists, we want to know more: So what do you do? You had…
Protein expression is an art. There are many routes to optimize a protein expression protocol, such as using different expression systems (e.g. E. coli, yeast cells, insect cells) or changing the expression vector or culture media for the expression host. Fortunately, optimizing the parameters mentioned above often leads to improvements in your protein expression results. However, there is one other…
Western blotting can often be a source of frustration in the lab. Getting a beautiful Western is hard work, and it’s even more difficult when trying to visualize large molecular weight (>150 kDa) proteins. Here are five tips you can use to get a great blot: 1. Choose the right gel composition With all of…
When you think about separating proteins, do you think about separating them using a gel? Specifically using SDS-PAGE? If you answered “yes”, it is for good reason. SDS-PAGE is ubiquitous in molecular biology labs because it is good at separating proteins. However, SDS-PAGE takes a lot of time and is labor-intensive. So let’s expand your…
If you were to peek into a protein biochemist’s bag of tricks, what would you find? A mortar and pestle for collecting samples, some columns for isolating proteins and a mass spec instrument? Perhaps. But what about those little eppendorf tubes full of enzymes and helpful molecules? Certainly, each scientist has his/her own favorite. Here…
If you are cooking up a way to test if two proteins interact, you need a yeast two-hybrid (Y2H) screen in your recipe book. You don’t want your Y2H to turn out half-baked – so check out this guide to Y2H and we’ll help you make sure yours will rise to the occasion! What is…
Problems with expressing your gene? One of the potential stumbling blocks in heterologous gene expression is incompatible codon usage. Every amino acid can be encoded by more than one codon, and for every amino acid each organism has a favorite codon that it tends to use more often than the others. The availability of tRNA…
Do you need your protein in its native form, intact, with full functionality? Do you need to isolate organelles and nuclear fractions from the cytoplasm? Or do you need a slurry of everything in your cell or tissue? Whatever your experiment, you can maximize the amount of functional, detectable or active proteins by handling your…
With in vitro translation (IVT) you can produce protein in a tube. Cool sure, but would IVT be a good choice for your experiments? Not sure? Then read on. Here I will cover the strength and weaknesses of IVT. How In Vitro Translation works First things first, for IVT you need ribosomes. While theoretically possible,…
You use your antibody frequently, maybe even every day. You rely on it for western blotting, immunohistochemistry, FACS, ELISA, and immunoprecipitation. You’d be lost without it. But how well do you really know your antibody? Are you sure that it detects what you believe it to detect? If you have the slightest doubt, or if you have…
It’s a familiar story – molecular biology meets protein science, they get closer and sparks fly. But how exactly does a proximity ligation assay (PLA) work and how do you make sure yours will have a happy ending? What PLA Does PLA allows you to detect individual proteins or individual protein interactor pairs without ectopic…
Bacterial cultures may be much easier to grow than mammalian cells, but if your yields are suboptimal there are plenty of parameters to play with. Here we list a few of the things you should consider to maximize your culture growth. Shaking speed Shaking is performed to allow aeration of your culture, which is of…
Chemical chaperones are necessary in protein experiments. From buffers to storage solutions, chemical chaperones silently make proteins happy and soluble. Read this article to appreciate the love that chemical chaperones bathe on your proteins and learn when to use them! A chemical chaperone is a molecule that promotes the favorable interaction of protein with water…
The half-life of a protein is an important factor in many molecular biology studies. If your thesis has anything to do with proteins then your graduate advisory committee will ask about half-life, so start planning your 35S experiment today. To help you here is my overview of the major steps of 35S labeling complete with…
Do you need purified recombinant protein to test biological drugs? But you don’t have the time or facilities for cell culture and purification? Try using in vitro translation (IVT) instead! IVT is like your own miniature protein factory. And it is great for a wide range of molecular biology applications because IVT can be much…
You’ve nurtured your cells for weeks, perfected your experimental conditions, and nailed down all the controls. You’ve harvested your cells and gently lysed them, now you’re ready to look at the proteins. What’s one of the most common next step in protein analysis? A denaturing gel or SDS-Polyacrylamide Gel Electrophoresis! SDS-Polyacrylamide Gel Electrophoresis, or SDS-PAGE…
Every protein is unique and thus every protein has its own set of production and purification challenges – many of which cannot be predicted. Therefore to successfully produce and purify your favorite protein you need to know and understand these five unpredictable protocol variables (X factors). Tweaking these X factors just might be the difference…
When it comes to Western blotting, there’s no denying it: Your membrane is a key player. After all it is the physical scaffold that holds your precious samples and it needs to be up to the challenges you throw at it. But depending on your protein’s properties and your downstream detection steps, finding the optimal…
Glucose is the preferred food source for E. coli, however when glucose levels drop, E. coli need to look for other ways to feed themselves. One way in which they accomplish this is to replace glucose metabolism with lactose metabolism. The induction and control of lactose metabolism is complicated and its process has been exploited…
Antibodies are one of the most important tools in molecular biology. Basic researchers use antibodies to identify, locate, isolate and quantify specific proteins. Clinical researchers use antibodies to target a specific drug (such as a chemotherapeutic agent) to a particular cell. Because of the cell specificity provided by the antibody, a higher amount of antibody…
If I piqued your interest in the first post about my new e-Book ‘The Bitesize Bio Guide to Protein Expression – a Bitesize Bio eBook’ check out this excerpt from the book explaining what an expression system is and how to choose the right one. What is an expression system anyway? There was a time…
An operon is a functioning unit of genomic DNA that contains a group of genes controlled by a single promoter. Put simply, these genes share information needed to create the tools for a particular task so they share a promoter ensuring they’ll all be transcribed together. The lac, or lactose, operon is found in E….
It has been said that “Incomprehensible jargon is the hallmark of a profession” (Kingman Brewster, Jr) and while I cannot speak for other professions, as a biologist I am inclined to believe it. So whether you need to cover your qualifying exam bases, want to avoid looking like an idiot to your coworkers, or need…
Westerns can be tricky and time-consuming, so make the most of your precious membranes and their proteins. Learn how to properly strip off your antibodies and re-probe with another primary antibody. Why you should strip Scientific reasons: To conserve protein samples that are limited or expensive. So that you can analyse the same sample with…
You have your favorite protein in mind and are ready to set up some exciting experiments to show what it does and how it does it, when it is active, what other proteins it modifies, and how it affects your cells. There is one slight problem. You need to fish your favorite protein out from the…
After you finish immunoprecipitating a protein or purifying a subcellular compartment, you need to identify what proteins you purified. You could attempt to identify your purified proteins the old fashioned (and slow!) way by running a multitude of Western blot. But rarely do labs have unlimited funds for Western blot antibodies. And lets face it,…
Blocking is the essential third wheel in any antibody/antigen relationship. Correct blocking buffer can perfect your antibody’s ability to bind its antigen, while bad blocking can make specific antibody binding near impossible. Don’t let bad blocking be a stumbling block in your Western blot experiments – read on to find out what blocking achieves and…
Radioactive protein labeling is not as common as it used to be. With the advent of modern protein labeling techniques, such as fluorescence, radioactive labeling has largely fallen out of favor. However, radioactive protein labeling is still a very useful technique and is often superior to more modern labeling techniques. Radiolabeling can provide a…
Perfect your subcellular fractionation in the lab by understanding how these protocols work. Plus, discover the various kits for organelle separation.
If you are struggling to optimise your Western blot protocol, one step to consider is the equilibration of your gel and membrane before transfer. Wondering what this step achieves and whether it’s necessary? You’re not alone! I did dozens of Westerns without ever bothering to equilibrate before I realised that it was having a big…
Stimulation of cells/tissue with a given stimulus (e.g., a cytokine) is a common experimental setup in any cell biology lab. The cellular response to the external stimulus e.g., the activation/deactivation of intracellular signaling pathways and/or the secretion of proteins is often the research goal, and there are a number of different methods that you can use to analyze such…
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