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Protein Expression and Analysis

Icky Sticky: Heat Shock Protein Contamination during Protein Purification

Purifying a new protein is no easy feat. Finding combinations of protein purification buffer, salt, detergent, and stabilizing agent to get high yields of squeaky-clean protein can become tedious. Few things are as bothersome during this process as Heat Shock Protein (HSP) contamination. But worry not, we’ve got some handy tips to avoid HSP contamination…

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Spinning Around: Tips and Tricks for Using Centrifugal Filters

One of the most widespread protein laboratory accessories are the MWCO (molecular weight cut-off) centrifugal filters which are commonly used for concentrating protein, as well as DNA. They are available commercially with different cut-offs including 3kDa, 30kDa, 50kDa, 100kDa, and so on. These little devices are expensive and hence demand proper usage and care to…

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Designing a LOV2 Domain-Based Toolkit

Cell Biology is entering the Age of Light with a spectrum of new optogenetics tools available to control protein function using light. Once the remit of neuroscientists [1], the past decade has yielded a bounty of novel light-controllable domains that are now being leveraged to illuminate the dark corners of basic cell biology [2,3]. The…

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So You Think You Can PEMSA? A Guide to Protein Electrophoretic Mobility Shift Assay

Studying nucleic acid interactions with proteins can be accomplished using a rapid and efficient electrophoretic mobility shift assay (EMSA). This method is essentially an agarose gel electrophoresis technique that detects protein:nucleic acid interactions, as the mobility of the labeled nucleic acid will be retarded if bound to a protein (compared to unbound DNA). A lesser-known…

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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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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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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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iPOND, Part 2: Why Should You Go Fishing with iPOND?

In part 1, ‘iPOND: Fishing for Proteins with DNA as Bait’ we went on a fishing expedition and learned how the iPOND technique can help us investigate the protein landscape of DNA synthesis. In this article, we will pit iPOND against a few other widely used techniques in order to answer the question: ‘Why should…

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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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iPOND, Part 1: Fishing for Proteins with DNA as Bait

No, iPOND is not a sleek electronic fishing device from Apple. However, if you thought about fishing, well, you’re not far off the mark. If you find yourself wondering which proteins are present at DNA during or after replication, iPOND is an elegant technique to help you find out. In this article, I will explain…

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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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Introduction to DREADDs – Control Over G Protein Coupled Receptor GPCR signaling

Gee, Protein, What Do You Do? Manipulation of a system under investigation is the backbone of experimentation. A new tool called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows us to hijack cell signaling and study cell function within living organisms. Like its cousin technique, optogenetics, DREADD technology uses a viral vector to introduce…

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A Primer on Phage Display Libraries

Phage display – the process of genetically fusing antibody fragments with phage to identify binding partners to your protein of interest – was covered pretty thoroughly here over the past few months. The success of this assay predicates on creating a diverse library of up to 1012 genes coding for these antibody fragments. Despite being…

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ELISA: How I Wonder What You Are

My phone’s email notification went off, and I rolled over in bed to look at the clock. Saturday, 5 am. Wonderful. Who would email me at that hour? It had to be my undergraduate research PI. I unlocked my phone. Yep. Doesn’t he ever sleep? Dear Casey: We are launching a new collaborative project in…

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Explained: Sensor Chips for Surface Plasmon Resonance and Other Applications

Biosensor chip selection is a critical step in planning and running a surface plasmon resonance (SPR) experiment. Chip selection depends on the ligand or target that needs to be immobilized on the sensor chip, the analyte that is flowed over the target to study the binding, and the purpose of the biosensor assay (i.e., determination…

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Development of a Potential Recombinant Protein Vaccine in E. Coli

Expression and Purification of an Engineered, E. coli-expressed Leishmania donovani Nucleoside Hydrolase with Immunogenic Properties Potential recombinant protein vaccine candidates must meet several criteria: They must be expressed at sufficiently high levels in the organism of choice They must be purified to high purity from the expression system in an immunogenic form They must induce…

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How to Store Your Concentrated Proteins

Like graduate students, proteins are sensitive to rough handling. This is particularly true when they (the proteins, not the students!) are being concentrated, purified, and stored. We’ve covered the many options out there for concentrating your proteins, along with how to handle protein extracts to keep your proteins safe from degradation. But proteins can degrade…

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How to Choose Quality Antibodies for Successful Western Blotting

Successful western blotting means achieving unambiguous results, and this requires a sensitive and specific antibody-antigen interaction. Consequently, high quality antibodies are critical for reliable and consistent western blotting. Western Blotting Process In the basic western blotting process, polyacrylamide gel electrophoresis (PAGE) separates a mix of proteins according to their molecular weights (denaturing gels) or their…

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