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Protein Expression & Analysis

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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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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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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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 Best Improve Your Lentivirus Titer

If you’re planning on using lentivirus for gene therapy, you will need to find out how many lentivirus particles to use. However, this is where researchers new to gene therapy work often run into problems. If this is you. Stop. Before you embark on endless optimisation experiments (i.e. a different transfection reagent, new cells, different phases…

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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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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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How to Make Your Own Luck in a Biochemistry Lab

During one of my first ever poster sessions, I was asked how I was able to make a particularly difficult-to-work-with protein soluble. My response was that I was just lucky. The venerable professor chuckled and stated that in science “you make your own luck.” This is a phrase that I have heard quite a bit…

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Isoelectric Focusing for Separation of Proteins and Peptides

SDS-PAGE is the standard technique used for separation of proteins in the lab, but that doesn’t meant that other techniques don’t have their place–one such technique is isoelectric focusing (IEF). IEF, also known simply as electrofocusing, is a technique for separating charged molecules, usually proteins or peptides, on the basis of their isoelectric point (pI),…

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How To Make Your Own ECL

ECL can be an expensive reagent in a lab, and what with it being involved in the final stage of a western blot, it’s something you don’t want to have to worry about too much. During my PhD, I was struggling with my western blots for ages – it seemed I was doing everything right,…

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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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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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