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Fluorescence is one of the most important and useful tools in a biologist’s toolbox. In biology, nearly every field, from physiology to immunology, uses fluorescent molecules (aka fluorophores) to detect proteins. However, the specific science behind how fluorescence works can be confusing or overlooked. Have no fear! In this article, we break down key points of…
In recent years, three-dimensional (3D) scanning electron microscopy techniques have gained recognition in the biological sciences. In particular, array tomography, serial block face scanning electron microscopy (SBFSEM) and focused ion beam scanning electron microscopy (FIBSEM) (described in Three-Dimensional Scanning Electron Microscopy for Biology) have shown an increase in biological applications, elucidating ultrastructural details of cells…
Grab an overview of CRISPR technology from its roots as a bacterial defense system to how it can be utilized in health and research.
Surface plasmon resonance (SPR) offers highly efficient, label-free detection for quantifying biomolecular interactions in real-time. Two exciting SPR variants that have sprung up in recent years are SPR for cellular analysis and SPR-mass spectrometry (SPR-MS). SPR for cellular analysis allows you to study how cells attach to different substrates and each other, while SPR-mass spectrometry…
Good quality starting material is king for reverse transcription! Obtaining reliable results in any experiment requires good preparation. We often take reverse transcription for granted, and we don’t always consider that our qPCR might be performing poorly because of problems in that step. Since it’s quite often the reverse transcription reaction itself that causes fuss…
Cryosectioning is difficult when your tissues melt, fold, curl, wrinkle, tear, or crack. Learn how to troubleshoot these pesky cryosectioning problems.
In this World of Microbes series, so far we’ve covered the importance of microbes in medicine including their use in vaccines, antibiotics, probiotics and protein production of antibodies for immunotherapy. Now we’ll diverge away from medicine and explore two other important fields where microbes are invaluable – food production and biofuel. Food Production When you…
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,…
Tissue culture can sometimes seem like a black art. Too careful—your cells go down. Not careful enough—your cells go down. A butterfly flutters its wings in the middle of the Atlantic Ocean—your cells go down. It’s annoying, it’s frustrating, and there are times (and I’m speaking from personal experience here) that you’ll end up chucking…
If you have sorted samples or phenotyped cells by surface expression of proteins, you’ve probably wondered how each cell is sorted or phenotyped in a flow cytometer? This question seems trivial, but in reality it took a while for engineers to figure it out. Before I get into today’s topic on “hydrodynamic focusing,” I’ll walk…
Viral vector production is a worthwhile skill that can be made even easier with a few tips and tricks. In general, transfection of multiple plasmids into a producer cell line results in infectious, non-replicative virus. However, it is important to ensure that your vector preparation is efficient, giving your experiments the best chance of success….
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…
There are several methods you can use to see if your T cells are cytotoxic, but a chromium release assay using radioactive 51chromium (51Cr) is one of the oldest. It gives good results, and is great for labs that can’t afford or don’t have flow cytometry readily available. Here, I will outline a simple method…
While the microscope is synonymous with biology, it is a child of physics and technology. When we learn about the microscope we learn physics—specifically, we learn about optics. Many great resources are available that explain the inner working of microscopy. And, like most things in physics, the inner working of microscopes comes down to a numbers…
Are you tired of staring at all of your sequence data? Want to know the easiest way to look at it? For complex genomics data, an appropriate visualization tool is a must have. The right genomics software will make it easy-peasy to get some results as well as test all those ideas you have. Since…
There are those of us who began our careers literally in the dark. Yes, there was a time and not that long ago, that all figures had to be on film. Slide presentations were slides. Micrographs were, well, micrographs on film. Figure creation involved several steps: figures for publications had to be mocked up; then…
Metabolomics may sound like a fictional character in the famous comic series “Asterix”, but it is very important in understanding systems biology and in clinical research against various diseases. Metabolomics is the last piece of a puzzle of omics applications following genomics, transcriptomics, and proteomics. It allows us to ask “What has happened and what…
So, you’ve designed PCR primers to amplify your sequence of interest, and you’re ready to go. But unless you have a never-ending supply of template, polymerase, and a thermocycler with a gradient function—not to mention a hefty dose of time and patience—you probably don’t want to spend the next week finding the perfect conditions for…
In Part I of AAV Production, I described how to produce crude (non-purified) AAV. In this article, I am going to tell you how to purify that crude prep. Virus purification is usually done by gradient ultracentrifugation. Two common methods involve gradients made from increasing concentrations of cesium chloride or iodixanol. A cesium chloride prep…
Multi-parameter data acquisition is key to the modern era of science research. I, for one, wish every single experiment that I design would give me the maximum amount of information. For example, in cell biology and immunology, we want to capture as much information (be it cytokines/hormones/chemokines) as possible about a given cell population. Of…
If you study human disease, you will likely handle a pre-analytical sample or two (or hundreds). For example, you could handle whole blood, serum or plasma, tissue biopsies, urine, fecal samples, cerebrospinal fluid, or synovial fluid—to name a few. You will probably use these samples to look for specific metabolites, proteins, or nucleic acids that provide…
Today, about half of all therapeutic drug approvals by the US Food and Drug Administration (FDA) are for biological drugs. That number is expected to rise to 75 percent by 2025. When these biologics come off patent, the gates will open for a flood of biosimilar drugs (biopharmaceutical generics) that are designed to be much…
An anti-cancer drug or antibody drug conjugate (ADC) screening assay is the first step to establish the utility of a drug candidate in killing cancer cells. Nevertheless, these assays are time consuming and tedious. The purpose of this article is to make things easier when you are required to set up these in vitro screening…
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…
When it comes to light sources for microscopy, there is really no such thing as the “best.” The type of light source you use depends on the system you are working with and the type of result that you want. Digital systems are usually designed to work with either a halogen or a LED light…
So, you’ve spent time planning your high-throughput sequencing experiment. You’ve chosen how many replicates to use, deliberated about sequencing depth, and kept everything RNase-free. Now you have many gigabytes of data available. What’s next? While the first step of RNA-Seq analysis is aligning your sequencing reads to a reference genome, first you need to get…
Welcome to the microbe series where we have a very exciting line-up planned over the coming months. Here we will talk about everything microbial, including the uses of microbes in industry and medicine, emerging pathogens, diagnostics, and much, much more! Let’s kick off this series with an introduction into these wonderful, yet sometimes nasty, organisms…
So, you have successfully cloned your gene of interest and are eager to purify buckets of protein. No matter your eventual application—kinetic experiments using a SPR instrument, structural analysis using X-ray crystallography, or any other experiment—you’ll need to express your protein first. Now, it’s time to put your expression plasmid into E. coli and get…
Surface plasmon resonance (SPR), a label-free, real-time way to examine protein binding and other molecular interactions, is getting easier as manufacturers have streamlined SPR instruments and supporting software. But problems can still arise. Troubleshooting Your SPR Assay Here are some common issues and suggestions to solve them: Inactive Targets Your target protein may have become…
If you have ever imaged biological samples, you have likely encountered autofluorescence. That pesky background coloration you see under the microscope, which can make it difficult to distinguish your actual signal from the noise.1 When you are trying to look for something as delicate as RNA, you don’t want to be hunting for your signal…
To many users, the flow cytometer is a magic box: put in cells, get out data. You click the button to tell it which colors to look at without much thought about how the machine does this. However, not all fluorophores are created equal—some configurations might exclude the spectrum you’re really looking for. Here’s a…
In austerity times, nothing is in excess. Apart from saving reagents, which can be refilled with extra financial injections, there is a commodity that cannot be easily resupplied – tissue samples! If, like me, you have experienced the fear of not having enough sample for performing a qPCR, western blot, and conventional PCR from the…
You already learned the basics of column packing. When moving to more automated system using low pressure liquid chromatography systems, you can use pre-packed columns. But in order to compare several resins in specific conditions, and also to save money, you might need to pack your own low pressure columns. The art of packing a column…
While using human clinical samples in your research can provide robust and heterogeneous results applicable to larger portions of the population, working with these samples presents its own set of challenges. Here are some tricks I have learned to help isolate and grow your cells of interest while eliminating stromal, blood, or other undesired contaminants….
So, the genome-wide association study (GWAS) data for your disease of interest was published, and it has thrown up some very interesting associations. However, at this stage, bear in mind that this is only an association. Your project is to provide the link between the GWAS single nucleotide polymorphisms (SNP) and pathological changes. Where do…
What is Adeno-Associated Virus? Adeno-associated virus (AAV) is a popular gene therapy vector. Different AAV serotypes infect different cell and tissue types with varying efficiency, so it’s a good idea to select the serotype most relevant to your work. The most commonly used AAV serotypes are 1, 2, 5, 6, 8, 9 and DJ, although…
Anytime lab processes get automated by a sophisticated scientific instrument, there can be a “black box” effect, leading users to wonder what’s going on in there. For DNA electrophoresis, it’s no different. It’s easy to see what’s happening in a manual gel, but the automated gel-based DNA size selection platforms can be more mysterious. Automated…
Are you preparing to set up live cell imaging experiments? You’ve got all your cell lines, antibodies, reagents, and protocol ready. You just want to wake up in the morning and enter into that dark room. Well, think again!! As we (I mean the cell biologists) always say, happy cells mean happy life. You have…
Running a pulsed field gel can be exciting. It isn’t often that you get to visualize such large pieces of DNA. However, it can also be extremely frustrating. It isn’t uncommon to wait 24 hours only to find out that your DNA was degraded before you started the run. Then, you have to start all…
Anchorage-independent assays test the ability of cells to grow independent of a solid surface. The assay is used to check the malignant potential of cancer cells. Cancer researchers generally do this experiment for any kind of confirmation of the oncogenic potential of an oncogene or a tumor suppressor in cancer cells. However, we do encounter…
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