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Posts Tagged ‘protein detection’

The 3 Most Common Flow Cytometry Fallacies

Flow cytometry is fast evolving from a method only revered by immunologists, to one used by nearly every biological specialty. It’s pretty much my favorite tool. Unfortunately, as with most lab techniques, much of flow cytometry is taught on the job without a lot of standards. And too often bad habits are passed along like…

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Fluorescent Western Blotting: Lowdown and Advantages

In this article, you will be introduced to the world of fluorescent western blotting. Firstly, we will compare fluorescent and chemiluminescent western blotting. Then, we will learn how infrared fluorescent western blotting can give you truly quantitative and reproducible results. Lastly, we’ll look at the many advantages of fluorescent western blotting, including the possibility to multiplex. Importantly,…

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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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Multiplex Cytometric Bead Array: The ABCs of CBAs

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…

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Four Tips for Working with Human Clinical Samples

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.…

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Lighting the Way: Understanding Flow Cytometry Fluorophores

As science is becoming more interdisciplinary, the tools we use to answer questions are also crossing party lines. Case in point: flow cytometry. Once a tool only used by “real” immunologists, flow cytometry is fast becoming a method by which numerous questions can be answered, from the length of a cell’s telomeres, to the state…

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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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Sonication – 7 Tips for Mastering the Art

Sonication is mostly used during preparation of protein extracts to help break apart the cell. Although most lysis buffers have buckets of detergent that lyse cell membranes, sonication just gives an extra hand in breaking everything apart. Sonication also breaks up, or shears, DNA in a sample—preventing if from interfering with further sample preparation. Have…

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Cell Cycle Analysis by Flow: DNA Stains and Beyond

While you can observe mitotic cell cycle progression using immunofluorescence, flow cytometry is a great tool to delineate details that aren’t apparent by chromosomal morphology alone. DNA stains are a great way to get a general idea of what your cells are up to. There are also a number of other stains you can use…

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The Messy World of Human Tissue Research

In neuroscience and other biomedical research fields, animal models allow us to answer critical but otherwise impossible questions. Despite the value of these models, however, sometimes nothing can replace the real deal—human tissue. The limitations of human tissue research stem from the variability between subjects and the risk for covariates that influence your results. Which…

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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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A Simple Method for Measuring Intracellular Fluorescence

Fortunately for microscopy users, measuring intracellular fluorescence has been made relatively simple through an ImageJ plugin called the Cell Magic Wand. For those of you unfamiliar with ImageJ, it’s a popular image processing program that runs on Mac, Windows, and Linux. How to use ImageJ for measuring intracellular fluorescence First of all, to begin measuring…

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Top 5 Tricks for Using FlowJo

Are you planning to do cellular immunology research?  Then chances are you will be introduced to the flow cytometer –  “a modern immunologist’s best friend.” This modern magic box is a highly versatile machine packed with cutting-edge fluidics and photonics (lasers). Combined with the monoclonal antibodies conjugated to fluorochromes capable of emitting light signals from a…

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Troubleshooting Thin Layer Chromatography: Some TLC for Your TLC

The whole TLC technique sounds easy to do, but it can be difficult and tricky during interpretation or give unexpected results, especially when working with biomolecules. For this reason, it is important to be familiar with troubleshooting thin layer chromatography. Some of the common problems faced during TLC and their solutions are listed below: Solvent…

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Mass(ively powerful) Cytometry

Mass Cytometry is a relatively new technology which has recently featured in many high-impact journals. You may have read about instruments including the CyTOF, CyTOF2, and more recently, the Helios. With these instruments becoming more widespread, you might find yourself asking, what is mass cytometry, and what can it do for you? The Basis: Conventional…

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Getting Started with Raman Spectroscopy: What You Need to Know

Are you an assiduous biologist who prefers label-free imaging methods for biological samples analysis? Raman spectroscopy offers you a wonderland of imaging technique with unlimited benefits. To start with, Raman Spectroscopy is a spectroscopic technique based on inelastic scattering of monochromatic light usually from a laser in the visible or near infra-red part of electromagnetic…

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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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Tips for Taking Immunofluorescent Images for Your Next Paper

Taking publication quality immunofluorescent images of can be a very time intensive, and frustrating process with hours spent capturing, processing, and putting the images into final figure format. And, if you aren’t careful, you can do a lot of work only to realize later that you need to re-image something for one reason or another.…

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How to See the Cell Cycle Through Your Microscope

Even in the most basic applications, fluorescence microscopy can be a very powerful technique. Simply put, the ability to actually see the biology you are interested in cannot be matched in directness. Often, the aim of fluorescence microscopy is to observe the effect of an experimental manipulation. Ultimately, you would like to know that the…

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Introducing Aptamers for Super Resolution Microscopy

What do you use if antibodies are too large for super resolution microscopy? Aptamers. These are small affinity reagents (~ 2 nm!) that interact with their target in the same way as an antibody, but without the hefty backbone attached.

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The Key to Unlocking DNA from FFPE Tissues

Formalin fixed paraffin embedded (FFPE) tissues are valuable samples that typically come from human specimens collected for examination of the histology of biopsies for the detection of cancer. But each sample contains much more information just waiting to be unlocked. Despite the tiny sample size, DNA can be extracted from the tissue sections and used…

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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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Immunoscience or Immunoalchemy?

First of all let me say the technique of labeling tissues (immunohistochemistry, IHC), and cells (immunocytochemistry, ICC) is indeed immunoscience NOT alchemy, though at times it may certainly seem like alchemy! But to scientists inexperienced in this technique, who typically see the results of IHC/ICC experiments in the form of pretty pictures, it can certainly…

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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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Remote Cytometry: Help from beyond!

The idea of accessing one computer from another is long established. Unfortunately, we often have visions of hackers sneaking in and stealing our data when we have most to lose. However, this type of technology can aid us in a lot of applications and to those of us who work in cytometry the benefits are (somewhat) clear. No More ‘Fail’ Moments Many researchers know the dread of…

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How to Make Lipid Bilayers

Supported lipid bilayers are a very useful tool in many fields of cell and membrane biology. But how easy is it to make them? Bilayers can be made quite reproducibly, once you have found a reliable protocol! However, it can take some time to optimize your technique, so to increase your chances, make sure you…

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