Protein Expression and Analysis
Bis-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 More How SDS-PAGE Works
Isolating 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 More 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.
Read More 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…
Read More 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…
Read More Nuclear Extraction: Protocol Mysteries Revealed!
Have you ever bought a kit from a biotechnology company and followed the instructions only to realize you’re not even sure why you’re doing certain steps in the prescribed way? I usually use a proprietary kit to perform nuclear extraction. A quick search on google for “nuclear extraction” will yield a plethora of kits for…
Read More Why Do Enzymes Have Optimal Temperatures?
Every biologist is familiar with the profile of the rate of an enzymatic reaction versus temperature as shown in the figure. We know that enzymes from E. coli or warm-blooded animals tend to have an optimum around 37°C, while those from thermal vent bacteria have much higher optimal temperatures. Surprisingly, I find that many biologists…
Read More 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.…
Read More 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…
Read More 2015 Staff Picks: Protein Analysis, Detection, and Assay
Today our artisanal article picks are from the Protein Analysis, Detection, and Assay. These picks are from our crack editorial team here at Bitesize Bio. After carefully combing through our archives, we have pulled these gems for your viewing pleasure. Jen Redig, Managing Editor How to Make a Custom Affinity Medium for Protein Purification by…
Read More 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…
Read More 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…
Read More 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…
Read More 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…
Read More 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…
Read More 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,…
Read More 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…
Read More Activity-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 More 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…
Read More 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…
Read More 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…
Read More 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…
Read More 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,…
Read More 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),…
Read More The Practical Guide to Running a Perfect Homemade SDS-PAGE Gel
As biochemists, we routinely run SDS-PAGE to analyze our proteins. It can be a repetitive and tedious task to get a “presentable” gel for publication (especially when there are Western blots to follow)… but not if you know the tricks!
Read More 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…
Read More The 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 More Overview 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 More Tips and Tricks for Western Blotting and Antibody Selection
You will learn: How to overcome common Western blot issues Which criteria to use for antibody selection Useful information for your grant preliminary data Western blotting is an important and widely used technique in cell and molecular biology. A Western blot is often used in research to separate and identify proteins based on molecular weight…
Read More Non-specific Binding? Tips to Sharpen up Your Western Blot
In the previous installment of this series on western blotting, we addressed potential sources of error when your final product is completely bare. But alternatively, what do you do when too much background is the problem? You may have beautiful bands of interest—but if there is a bunch of non-specific binding, your quantification and data…
Read More Critical Success Factors for Sample Collection, Storage and Retrieval
In this webinar you will learn: How to maintain specimen integrity when storing tissues, blood and cells. How to store and retrieve specimens from a biobank. Special considerations for preparing formalin-fixed paraffin-embedded (FFPE) tissues for storage. Abstract: Biobanks, or biorepositories, store biological samples that are critical for research in the biomedical fields. Samples from biobanks…
Read More Streamline Your Western Blots
Western Blotting is a long established method for which the protocol varies little from lab to lab. However, there are some new products that are available and some tweaks that can be made to the protocols that may improve your results and reduce the time it takes you to execute this popular technique. Save Time…
Read More How to Optimize Your Western Blot Transfers
So, you’ve done your experiment, prepped your samples, and run your SDS-PAGE gel. Now it’s time for the all-important transfer step, that tricky point that will determine the quality of your Western blot. Transfer times are empirical and based on your own particular samples, which means that there is no easy way to determine how…
Read More 8 Basic Points to Remember Before Expressing Proteins in Bacterial Systems
As a protein biochemist and a Ph.D. student, I was given the task to express a eukaryotic protein in a bacterial system. And to say that I was having a hard time would be an understatement. It took me many PCRs, cloning and transformations to get to the right construct that would eventually express the desired…
Read More 11 Reasons Why You Should Use Recombinant Antibodies (rAbs)
Monoclonal antibodies: You’ve probably heard a lot about them. Unsurprisingly, you may have also used them in your research. These antibodies (mAbs) are classically produced by the hybridoma technology pioneered by Köhler and Milstein in 19751: A mouse is immunized with the substance against which you need to produce an antibody. The mouse spleen cells (consisting…
Read More Roadside Assistance: Fixing Your Broken-Down ELISA
The ELISA (enzyme-linked immunosorbent assay) is arguably one of the most important and versatile tools in the toolbox of molecular biologists, biochemists and diagnosticians across the world. Defined by its simplicity and speed, the assay is easy to learn and perform in as few as five steps. But with so few variables to manipulate, an…
Read More Express yourself: Gene Co-Expression in Bicistronic Constructs
Let’s say you work with a gene, and it has wonderful potential. Excitedly, you throw your gene into the cells and voila! It’s there. Great. Now what? Genes are powerful tools for directing cell activity, but thanks to that curiosity characteristic of scientists, we want to know more: We want to be able to confirm…
Read More The Art of Protein Expression – How Changes in the Universal Genetic Code Can Affect The Activity of Your Expressed Protein
Protein expression is an art. There are many routes to optimize a protein expression protocol, such as using different expression systems (e.g. E. coli, yeast cells, insect cells) or changing the expression vector or culture media for the expression host. Fortunately, optimizing the parameters mentioned above often leads to improvements in your protein expression results. However, there is one other…
Read More Five Fast Tips for Blotting Large Proteins
Western blotting can often be a source of frustration in the lab. Getting a beautiful Western is hard work, and it’s even more difficult when trying to visualize large molecular weight (>150 kDa) proteins. Here are five tips you can use to get a great blot: 1. Choose the right gel composition With all of…
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