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Protein Expression & Analysis

Crash Course in Fourier Transform Infrared Spectroscopy

Fourier transform infrared spectroscopy (FTIR) is an extremely sensitive technique for measuring the absorption and intensity of electromagnetic radiation in the infrared region of the spectrum of either a solid, liquid or gas sample. You can use FTIR to: quantify unknown compounds identify unknown compounds study the detailed structured coordination of compounds How Does Fourier…

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An Introduction to Chimeric Antigen Receptors (CARs)

You may have heard about a breakthrough cancer therapy that engineers patient’s immune cells to fight their cancer using  chimeric antigen receptor (CAR)-T cells. If you don’t live in the world of immunology, you may not know what a CAR is, or what it is used for. Here you’ll find a brief guide to CARs,…

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Fishing for Kinases with Multiplex Inhibitor Bead Assays

There is something about kinases that resemble ghosts. Their effects reveal their presence, but they can be difficult to catch. With a low abundance of hundreds or even tens of molecules per cell, they are difficult to detect using conventional methods such as Western blotting or mass spectrometry (MS). However, you will need to detect…

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Top Five Methods for Primary Antibody Labeling

In any application that uses antibodies for signal detection (e.g., Western blotting, ELISA, immunohistochemistry, or FACS), there are two approaches to antibody labeling: direct and indirect labeling. Standard Western blotting uses indirect labeling because you use a primary antibody to detect the target antigen, followed by a secondary antibody to which a detection molecule is…

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An Experimental Tool-kit for Measuring Protein Stability

Proteins in the cell are in a constant flux governed by events including synthesis and degradation. In an effort to make cells more efficient by reducing the unnecessary protein load, most proteins in the cell have a specifically defined half-life. Another reason why cells have evolved to degrade proteins is to ensure timely removal of…

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Helpful Tips Before Your First Rho Pull-Down Assay

If you have studied cellular movement or cell division, you have encountered Rho in the literature, because it regulates both processes. And the list of roles for Rho in the cell continues to grow! The prominence of Rho in the biology of non-diseased and diseased cells has caused researchers to continually optimize the Rho pull-down…

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Troubleshooting a Faulty ELISA

Is ELISA giving you the blues? The frustrating kind, not the lovely kind you get while watching the enzyme substrate reaction! This age old assay has the perks of being quick and fairly simple to perform, but when conditions are not perfect, ELISAs can deliver less than optimal results, and fail to be consistent and…

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Protein Self-Labeling with Halo, SNAP, and CLIP Tagging

We all know the impact fluorescent proteins have had in advancing cell biology. Although fluorescent proteins have revolutionized the field, they aren’t perfect and like all things research, they have their limitations. If you’re looking for a genetic tool with superior fluorescent properties, or one that allows you to introduce a variety of labels into…

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Quantifying Individual Proteins Per Bacterial Cell

I’m a simple molecular biologist. It’s awesome how computational biologists use math to reduce and rebuild biological phenomenon. In my own way, I also like to reduce my observations to numbers. As a budding biochemist, I need to assemble and quantify the players in my pathway to truly understand it. In particular, I am interested…

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Modeling – in the World of Proteins

Computational protein structure prediction provides three-dimensional structures of proteins that are predicted by in-silico techniques. Such protein modeling relies on principles from known protein structures obtained via x-Ray crystallography, NMR Spectroscopy, as well as from physical energy functions.  There are three main methods of modeling: The first and favorite method is Homology Modeling,1-2 Followed by the…

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An Introduction to In-Gel Zymography

Enzymes are special among proteins. It is not enough to detect them. You need to know their activity level. If you have devoted a substantial part of your research to studying proteases, like I did, you’ll know how crucial it is to choose an appropriate enzyme assay. There’s a heap of lab techniques out there…

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How to Prepare Biological Metallo-Proteins

The first thing one might notice when working with metallo-proteins is that they offer unique, colorful reactions.  These colorful reactions are based not only on the metal, but the ligand, or coordinating molecules.  Approximately 80% of proteins contain inorganic cofactors like iron (Fe) and copper (Cu) metals necessary to catalyze a reaction.  Understanding how these…

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Brefeldin A v Monensin: How to Hunt for Proteins

As any good biologist knows, one of the easiest ways to determine if a cell is functionally active is the production and secretion of proteins in response to a stimulus. In many circumstances, the quantity of the secreted protein, and thus the level of cellular activation can be assessed by ELISA. However, if you are…

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Taking up the Challenges of In Vitro Monoclonal Antibody Production

Monoclonal antibodies are extensively used in research laboratories, diagnostic products and immunotherapy and have multiple advantages over polyclonal antibodies. They exhibit enhanced specificity to single epitopes, have little or no variability, and are easy to modify and customize as required. The History of Monoclonal Antibodies In 1984, Georges Köhler, César Milstein, and Niels Jerne received…

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Codon Optimization for Increased Protein Expression

The Genetic Code: A Universal Template for Protein Translation All known organisms share the ‘central dogma’ of molecular biology. DNA is transcribed into mRNA that is translated into protein. During the discovery of the genetic code, Francis Crick hypothesized that translation required a mediator to aid mRNA-guided translation according to a number of specifics. Amongst…

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Antibody Validation

Not all antibodies are valid for every experiment and condition, and they must be validated for the specific application and species. Currently, there is no standard means of “antibody validation,” and this can greatly impact experimental reproducibility and reliability. Journals and granting agencies have taken steps to address this gap. Many now require you to…

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