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Protein Expression and Analysis

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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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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Fluorescent Western Blotting: Lowdown and Advantages

In this article, you will be introduced to the world of fluorescent western blotting. Firstly, we will compare fluorescent and chemiluminescent western blotting. Then, we will learn how infrared fluorescent western blotting can give you truly quantitative and reproducible results. Lastly, we’ll look at the many advantages of fluorescent western blotting, including the possibility to multiplex. Importantly,…

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Molecular Docking: Let the Docking Do the Talking

Want to know whether a lead molecule or a ligand of your choice interacts with a particular protein or a receptor? Do you want this information at your fingertips? All it takes is just a few clicks and key presses on the computer and then out comes a computational prediction. This computational process is also…

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Titering Phage – Counting Something Invisible with Something Only Slightly More Visible

Titering Phage – The Plaque Assay Phage display is a molecular technique used to isolate binding or interaction partners to molecules of interest from an extensive library. Such libraries are often derived from the variable regions of native B-cell antibody-binding genes cloned into phage DNA. A single round of phage display panning involves many important steps. However, the…

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Proteomics and Good Mass Spectrometry Data

It is currently possible to analyze thousands of proteins in a single sample using mass spectrometry (MS) and a database of predicted protein sequences, referred to as ‘bottom-up’ proteomics. With this technology, you can measure protein levels and interactions. Also, you can examine changes in post-translational modifications (PTMs) and isoforms (in an unbiased manner). Working with…

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