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Protein Expression and Analysis

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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Buffer Banter: Pre-cast PAGE Gels & Buffer Compatibility

This article is not for the die-hard old-school gel runners. You know who you are, the purists, the “I always make my own gels and buffers from scratch” kind. For you we have lots of articles about PAGE gels, both bis-tris and the standard SDS PAGE kind. Instead this article is for the rest of…

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Extracting Better Ubiquitin Data from Your Samples: Beyond the Cellular Skip

The ubiquitin-proteasome system was discovered at the start of the 1980s, and people have been studying it ever since. Initially, researchers thought that tagging a protein with ubiquitin was the cell’s signal for the protein to be scrapped via the proteasome. But more research has shown that, as with all biology, once you’re up close and…

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A New Frontier in Protein Quantitation: AlphaLISA

If you ever worked in a biology or biochemistry laboratory, you probably already heard about ELISA. You may have even used it. But do you know what’s behind it? And how you can improve it? Let me guide you through the basics of ELISA, and introduce you to my favorite ELISA technique—AlphaLISA. First Things First… So,…

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How to Scrutinize Your Glycosylated Proteins Without Using Glycosidases

You might have come across protein glycosylation before. Somewhere in the recesses of your memory you might even recall reading something about the protein you’re studying being glycosylated, but what does this mean and how do you analyze it? Glycosylated proteins are molecules decorated with sugar groups as they pass through the ER and Golgi…

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Key Analytical Challenges for Antibody Drug Conjugates

Currently, there are more than 75 antibody drug conjugates (ADCs) in various stages of pre-clinical and clinical development. The combination of a targeted antibody coupled with a cytotoxic small-molecule drug (via  a flexible linker) makes for a lethal and specific oncologic drug product. However, an ADC is a heterogeneous cocktail of molecules with a range…

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Ten Ways to Give Your Surface Plasmon Resonance Experiments a Hand

Surface Plasmon Resonance (SPR) is the gold standard for measuring biomolecular binding without the need for labeling (i.e., label free detection of kinetics). SPR is especially valuable because it doesn’t just provide information at the start and end of a binding event, but can be used to follow association and dissociation kinetics of biomolecules in real-time.…

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Have a BLAST With Your Protein Sequences

When I was being trained in microbiology as an undergrad, one of the first skills I acquired was the ability to quickly compare and visualize amino acid sequences using BLAST and ClustalW. 15 years later, those two programs have done nothing but improve by expanding the data contained in these databases and simplifying the user…

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Stripping Blots – It’s All Fun and Games Until Someone Loses Their Protein

Like all technical fields, molecular biology contains a very robust “theoretical” realm and an equally robust “practical” realm. Unfortunately, these two existences don’t seem to overlap as often as we’d like. Consider, for example, a simple Western blot. While an antibody interacting with its target on a membrane seems pretty straightforward, there are numerous other…

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How to Cherry Pick Your Primary Antibody

How do you pick which antibody you should use in your assay? If you’re starting a new assay and need an antibody for the job, then selecting a new antibody from the plethora available could be high up on your to-do list.

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A Beginner’s Guide to Tag-Removing Proteases

Every biochemist is familiar with proteases. More often than not, proteases cause a lot of anxiety. To this end, a lot of research has been done in developing techniques to prevent the activity of proteases. But some of these proteases can be the good guys too! For example, you can use them to separate your…

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How to Use Proteases to Purposefully Digest Proteins

In this article I will not talk about ‘wild’ proteases, which destroy cellular proteins in your lysates like wolves destroy sheep. Instead, I’ll be talking about the shepherd dog proteases—purified, tame and useful to digest proteins your research. In Protein Research and Crystallization Several programs can predict your protein domains. However, we wet biologists know…

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Native Versus Denaturing Gels

We’re already gone through the basics of how gel electrophoresis work, compared common gel types like agarose and polyacrylamide and even explored some alternatives. Now let’s look at the native versus denaturing gels. You’ll be a speGEList in no time! Denaturing Gels We’ll start with this one, as it’s very self-explanatory. Denaturing gels are exactly…

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Go Fishing for RNA-Protein Interactions with a Yeast Three-Hybrid Assay

If you’re hoping to reel in a positive interaction between a protein and an RNA sequence, try to catch a winner with a yeast three-hybrid assay. What is yeast three-hybrid (Y3H)? The Y3H system is based on the same principle as a yeast two-hybrid– namely, that the DNA binding domain and the transcription activation domain…

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Finding a Needle in a Needlestack Using Phage Display

Few things can dash your hopes quite like phages. They can annihilate whole bacteria cultures in the blink of an eye, and make your next cloning or expression project impossible. But you can harness these evil-do-ers for good. And use phages to screen massive libraries of peptides. Learn how below. The Typical “Evil” Phage Experience…

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An overview of the Yeast one-hybrid assay

If you are regularly doing ChIP-qPCR, ChIP-RNAseq or luciferase reporter assays to measure protein-DNA interactions, then this article is for you! ChIP experiments can tell you what DNA sequences your protein binds, and luciferase reporter assays predict whether your protein functionally binds a specific promoter to activate transcription – but a yeast one-hybrid (Y1H) assay…

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The Lab Detective: Finding the Right Blot Detection Method

When it comes to registering the signal output of your Southern/Northern/Western/probe hybridization, you are spoilt for choice these days. You can go all retro and use X-ray film. You can go digital and use a phosphorimager. Finally, you can go fluorescent and use a fluorescence detector. So, what are the pros and cons of each…

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Protein Extraction and Solubilization using the TRIZOL® Method

Extracting protein from tissue samples and cultured cells is Step #1 in many biochemical and analytical techniques. Before you can do a Polyacrylamide Gel Electrophoresis (PAGE), a Western blot, or mass spectrometry you need to extract your protein. Nowadays, a lot of labs have switched to kits for their protein extraction but these kits can…

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How to Best Improve Your Lentivirus Titer

If you’re planning on using lentivirus for your next experiment, chances are you’re wondering how much virus to use. For in vitro work, multiplicity of infection (MOI) is the theoretical number of virus particles applied per target cell. That is to say, if you have 1 million cells and you want an MOI of 5,…

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How to Express an Elusive Protein

As a research intern this summer, part of my project included expressing and purifying a few proteins of interest. Two out of the three proteins posed no problem, but the third caused me to spend an agonizingly long amount of time– setting up new secondary cultures everyday, waiting for them to grow, forgetting to induce…

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Where are My Bands? Troubleshooting a Signal-less Western

Western blotting uses electrophoresis and antibody-epitope affinity to give a semi-quantitative and (theoretically) clear measure of protein abundance. It’s a long procedure, filled with many steps—and even more room for error. Learning to troubleshoot certain problems is incredibly important for continued success with this technique. So what do you do when your final imaged product…

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How to Shut Off Background Lac Expression in LB

Here’s a tip that you may find useful if you are expressing proteins in E.coli using a lac promoter-based expression system, e.g. pET, in LB medium (L-broth). Lac expression systems are typically induced in the lab using IPTG (isopropyl-beta-D-thiogalacto- pyranoside), which is a non- hydrolyzable analog of lactose, the natural inducer of the lac operon.…

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Origami in Nature: Protein Structure Prediction

Predicting how proteins will fold in vivo is a Holy Grail of proteomics and theoretical chemistry. Current hopes are that this can be achieved by designing an in silico platform that can predict protein folding, either de novo (a.k.a. from scratch) or using known proteins as a guide. What would we need to do, why…

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Gain Control: The Tet-On/Tet-Off Inducible Expression System

While overexpressing a gene of interest can provide a look into its role in a cell, sometimes it is necessary to control the expression of a gene. You may want to dictate the timing of the protein’s expression or lower its expression level to adequately understand its function. This is particularly relevant when studying genes that…

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How to Make a Custom Affinity Medium for Protein Purification

Is your goal to purify a substantial amount of a specific protein? Do you have a quantity of a molecule that binds your protein of interest? If so, generating a custom affinity matrix may be just the trick you need to purify your protein of interest by affinity chromatography. Customizing your affinity chromatography is an…

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Breakthroughs in Peptide Translocation: Cell Penetrating Peptides

Cell Penetrating Peptides (CPPs) are the Trojan Horse of cell biology – an innocuous peptide sequence with the special ability to carry virtually any cargo across the plasma membrane. If you have a special delivery that you don’t want to get lost in transit, CPPs are for you! CPPs are short peptides (typically 5-30 amino…

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How To Make Your Own ECL

ECL can be an expensive reagent in a lab. Why not make your own? Hopefully, this quick, simple and cheap solution will be of help to you!

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Split Ubiquitin Yeast Two-Hybrid

If you’ve read our article, An Overview of Yeast Two-Hybrid (Y2H) Screening, you’ll know that one major limitation of conventional Y2H is that your protein-protein interaction must occur in the nucleus for the reporter gene to be activated. So what do you do if your protein is a receptor tyrosine kinase? Or a G protein–coupled…

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How Sweet is Your Protein: Using Enzymes to Study Glycosylation

Most eukaryotic proteins exist as several isoforms, differing in posttranslational modifications, which allows them to perform slightly different functions or the same function under slightly different conditions. A common posttranslational modification of proteins is glycosylation.

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