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Protein Expression and Analysis

The Mysterious World of Macrophages: How to Harness Them for Your Research

The Mysterious World of Macrophages: How to Harness Them for Your Research In this tutorial, you will learn: How to work with mouse and human macrophages The best methods for culturing macrophages The difference between M1 and M2 macrophages, and how to differentiate each population from monocytes What data to expect based on the conditions…

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How it Works: Storage Phosphor Screen

Radioactivity is still the most sensitive detection mechanism for many macromolecules and enzymatic activities. In graduate school, I performed countless radioactive kinase assays, watching the radioactive gamma 32P of ATP get transferred to my autophosphorylating receptor of interest, and then separating my protein from free hot ATP on a gel. The gel is dried, covered…

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3 Ways to Use Flow Cytometry for Your Activation Experiment

Studying immune cell activation allows scientists to understand the way the body mounts a response to a specific infection, autoimmune diseases, or cancer. This knowledge plays a direct role in developing more efficacious vaccines and therapies. When tasked with capturing information on immune cell activation, flow cytometry remains the gold standard due to its versatility,…

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An Exploration of the Sigmoidal Curve – Math for the Rest of Us

ELISA (Enzyme-Linked Immunosorbent Assay) is the heartbeat of many labs in the research world, owing to its simplicity and its ability to answer a very basic question: how much of protein/peptide/antibody is in my sample?  More specifically, it can be used to answer such questions as: How much IgG is in the serum after I…

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Will the Real NP-40 Please Stand Up? Chemical Nomenclature Woes.

One day, a colleague stopped by my workbench to ask which detergent would not break the nuclear membrane. Based on my previous experience using gentle detergents in lysis buffers, I replied, “NP-40”. However, we had two brands of NP-40. A closer look at the datasheets revealed that the chemical names were different even though they…

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How Protein Adhesion Can Affect Your Experiments and What to Do About It

Commonly, no one thinks much about how the surfaces of labware (like microcentrifuge tubes, test tubes, dishes, etc.) can affect experimental results. We might know when we need to use glass versus plastic. Or we might know that certain chemicals, like chloroform, will interact with some plastic polymers, and you must use polymers that are…

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Five Methods for Assessing Protein Purity and Quality

If you’ve ever worked with proteins in the lab, you probably know just how critical protein purity and quality are for downstream applications. In this article, we’ll review the multitude of problems that are encountered with ‘bad’ protein samples and how you can analyze the purity and integrity of your favorite protein prior to using…

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Let’s Come Clean: Why Quality Control for Purified Protein Matters

If you are a Michelin star chef, then your first priority for preparing your signature dish is to use the best ingredients. One rotten potato or one slightly overripe strawberry could ruin not just a dish, but also your reputation. In the laboratory we are (mostly) not cooking rotten potatoes, but we are doing delicate…

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8 Tricks to Improve Your Negative Staining of Membrane Proteins

Negative staining of proteins is a versatile tool for structural biology. The sample preparation protocol is simple: the sample is embedded in a heavy metal stain that gives rise to increased specimen contrast. Thus, negative staining is a very convenient method to assess sample homogeneity, formation of macromolecular complexes, or quality of protein preparation. Conventional…

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A Beginner’s Guide to Measuring Metabolism In Vivo

Have you ever seen a mouse chowing down on its dinner and wondered how it translates to energy? Well, that’s exactly what is keeping some scientists up at night, and luckily these questions are now becoming easier to answer in quite significant detail. Over the last couple of decades, several companies have developed ‘metabolic cages’.…

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Crash Course: Developing a Fool-Proof ELISA

Crash Course: Developing a Fool-Proof ELISA Join us in this webinar featuring Dr. Omonse Talton who will guide you through developing a “fool proof” enzyme-linked immunosorbent assay (ELISA).In this tutorial, you will find: When and why you should use the different types of ELISAs– direct, indirect, sandwich, competitive/inhibition ELISA Major considerations for developing your ELISA…

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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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