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…
Read MoreTitering 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…
Read MoreProteomics 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…
Read MoreBuffer 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…
Read MoreExtracting 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…
Read MoreA 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,…
Read MoreHow 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…
Read MoreKey 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…
Read MoreTen 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.…
Read MoreHave 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…
Read MoreStripping 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…
Read MoreSDS-PAGE Tips and Tricks to Save You Time
Speed up your SDS-PAGE with our time-saving tips and tricks!
Read MoreHow 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.
Read MoreA 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…
Read MoreCommon Mass Spectrometry Contaminants: I Messed It Up So You Don’t Have To!
Through many trials, and lots of error, I learned that there are many considerations for mass spectrometry that might not be obvious to you as a molecular biologist. Common contaminants, even in small quantities, can mask important peaks in your mass spec data and have a huge impact on the final results.
Read MoreHow 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…
Read MoreSoluble Sample Success: How to Optimize Protein Solubility
Put in the time now to optimize protein solubility so you don’t have to suffer later! With this in mind, here are some considerations and tips for selecting the optimal conditions for recombinant protein expression and purification.
Read MoreBis-Tris Gels: Sharpen Up Your Protein Bands
A sprinkling of Bis-tris is all it takes improve your protein gels considerably. If you can afford it!
Read MoreIsolating Proteins? Use Histidine and Transition to Metals
To isolate your protein, you are going to need to tag it with something that will allow you to fish it out of the bacterial protein soup. This is where transition metals can help you out!
Read MoreOverview of In Planta Sub-Cellular Localization by Agroinfiltration
Agroinfiltration is a method for the transient expression of your protein of interest in a plant system. You can use it for the production of recombinant proteins or simply to determine the sub-cellular localization of your protein.
Read MoreNative 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…
Read MoreGo 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…
Read MoreFinding 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…
Read MoreAn 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…
Read MoreThe 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…
Read MoreProtein 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…
Read MoreHow 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,…
Read MoreHow 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…
Read MoreWhere 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…
Read MoreActivity-Based Protein Profiling: A Powerful Technique for the Modern Biologist
Imagine trying to build a house without power tools: It’s completely doable – after all, people did it that way for centuries – but it’ll take you a lot longer and has limits. Similar to this, modern day biology now has its own set of “power tools.” So while you could do biology the old-fashioned…
Read MoreHow 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.…
Read MoreOrigami 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…
Read MoreGain 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…
Read MoreHow 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…
Read MoreBreakthroughs 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…
Read MoreHow 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!
Read MoreThe Wet-chamber Method: How to use Less Antibody and Save Money
Want to save money on one of the most expensive steps of your Western blots? Then, use less antibody for significant cost savings!
Read MoreThe Practical Guide to Running a Perfect Homemade SDS-PAGE Gel
As biochemists, we routinely run SDS-PAGE to analyze our proteins. Imagine the time and effort you are going to save when you can run every gel to perfection.
Read MoreSplit 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…
Read MoreHow 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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