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Protein Expression and Analysis

Immune Responses in Severe COVID-19 Patients

Immune Responses in Severe COVID-19 Patients On Demand Speaker Dexi Chen Professor and Director Prof. Dexi Chen is the Director of the Beijing Institute of Hepatology since 2010, mainly focused on molecular and cellular biology of infectious diseases, and the mechanisms of hepatocarcinogenesis induced by HBV. Previously, Prof. Chen led the HIV research lab at…

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Assay Development for Quantification of Low-abundance Proteins Using Single Molecule Counting

Assay Development for Quantification of Low-abundance Proteins Using Single Molecule Counting On-Demand Sponsored by Sarah HamrenMilliporeSigma In this tutorial you will find: How MilliporeSigma’s Single Molecule Counting (SMC™) platform can be used to detect even very low levels of Huntingtin protein How to reach fM levels of sensitivity detecting both mHTT and total HTT Development…

Neurons, showing how Single Molecule Counting can be used for detection of Huntingtin protein for Huntington’s diseaseRead More

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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Laemmli Buffer: What Is It for Anyway?

Electrophoresis encompasses a wide range of techniques in which charged biomolecules in a liquid, a solid, or a semisolid solution can be separated by size under the application of an electric field. The most common application of electrophoresis for the separation of proteins is SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), which has been previously covered here.  …

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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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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…

bubble and squeaky cleanRead More
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