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Search below to delve into the Bitesize Bio archive. Here, you’ll find over two decades of the best articles, live events, podcasts, and resources, created by real experts and passionate mentors, to help you improve as a bioscientist. Whether you’re looking to learn something new or dig deep into a topic, you’ll find trustworthy, human-crafted content that’s ready to inspire and guide you.
Blocking is the essential third wheel in any antibody/antigen relationship. Correct blocking buffer can perfect your antibody’s ability to bind its antigen, while bad blocking can make specific antibody binding near impossible. Don’t let bad blocking be a stumbling block in your Western blot experiments – read on to find out what blocking achieves and…
Radioactive protein labeling is not as common as it used to be. With the advent of modern protein labeling techniques, such as fluorescence, radioactive labeling has largely fallen out of favor. However, radioactive protein labeling is still a very useful technique and is often superior to more modern labeling techniques. Radiolabeling can provide a snapshot…
Perfect your subcellular fractionation in the lab by understanding how these protocols work. Plus, discover the various kits for organelle separation.
If you are struggling to optimise your Western blot protocol, one step to consider is the equilibration of your gel and membrane before transfer. Wondering what this step achieves and whether it’s necessary? You’re not alone! I did dozens of Westerns without ever bothering to equilibrate before I realised that it was having a big…
Stimulation of cells/tissue with a given stimulus (e.g., a cytokine) is a common experimental setup in any cell biology lab. The cellular response to the external stimulus e.g., the activation/deactivation of intracellular signaling pathways and/or the secretion of proteins is often the research goal, and there are a number of different methods that you can use to analyze such…
You spent the last few weeks tweaking your Co-immunoprecipitation conditions, testing different antibody/bead combinations, and sampling a panaply of solutions and FINALLY! You have your Co-immunoprecipitation (Co-IP) elution… Now what? Well, you have a few choices. It really all depends on what you need know about the proteins in your elution. Do you need to identify…
Ever had a blot so bad it looked like a Rorschach test? We have ten things that might be going wrong with your western blots and how to fix them.
Do you wonder if your favorite protein interacts with another protein? Do you wish that you could shine a spotlight on your protein to determine its binding partner? You can use co-immunoprecipitation (Co-IP) to find your protein’s partner. This article will get you ready for your first Co-IP, provide a handy Co-IP protocol, and discuss…
Western blots can be ugly. I mean down-right, horrifically, wall-of-shame ugly. Not only can they be embarrassing to show to your colleagues, but the ugliness can obscure your results, making it impossible to interpret your data. Blotting consists of many experimental steps, which makes the technique naturally error-prone. Although standardized protocols exist, many fail to…
An ELISA (Enzyme-Linked ImmunoSorbant Assay) is a popular assay that uses antibodies and color change to detect proteins, peptides, antibodies or biomolecules in complex mixtures. ELISAs are popular because they are reliable, specific, easy to use, and can easily be scaled up to process multiple samples simultaneously. How an ELISA is Done: In an ELISA,…
Isoelectric focusing electrophoresis (IEF) of proteins is nowhere near as popular as its cousin – sodium dodecyl sulphate-polyacrylamide gel electrophoresis aka SDS-PAGE. While in both methods the proteins are denatured, IEF is a gel-based electrophoretic separation of proteins using difference in their overall charges. The sodium dodecyl sulphate – SDS part of the usual gel…
Wouldn’t it be great to put your nucleotide sequence into a program and get back a 3D-structure of your protein and a full description of its functions? In theory, because the protein 3D-structure is determined by the aminoacid sequence, given the right algorithm and a powerful enough computer, this should be simple. In practice, because…
Have you ever emerged from the lab, bleary-eyed, blinking dazedly at the sun after spending hours hunched over a lab bench counting endless bacterial colonies or viral plaques? A necessary evil… I consider colony/plaque counting one of the necessary evils of working with microorganisms. Necessary because many experiments have an endpoint that requires determining the…
To answer some of the more interesting research questions, you often need to get a good look at what’s going on inside the cell. Whether you’re running a Western blot or measuring enzyme activity, many assays require access to the materials (e.g. proteins, DNA, subcellular fragments) contained within the cell walls. There are several ways…
In the previous article on EC numbers, I explained how the Enzyme Commission names enzymes, and why it is so important. In this article I’d like to take you on a brief journey through the history of the Enzyme Commission. Like many histories in science (e.g. this!), it is fascinating and gives a useful perspective…
As a biologist you will no doubt have seen Enzyme Commission (EC) numbers. An EC number is group of four numbers separated by periods in papers discussing enzymes…something like this: EC 1.1.2.1. But do you know what these numbers mean? Or where they came from? Or why we use them? If not, I will aim…
The ELISA (enzyme-linked immunosorbent assay) is a rapid method used to detect the amount of a protein of interest in clinical and experimental samples. There are a number of ELISA formats to choose from, depending on your research needs. These include direct ELISA, indirect ELISA, competitive ELISA and sandwich ELISA. We have previously covered the…
In her article How to Get Perfect Protein Transfer in Western Blotting, Emily Crow recommends Coomassie staining your gel after transfer to the membrane to check the quality of the transfer. A good transfer should not leave behind proteins and PVDF membrane, stained by 0.1% Ponceau S in 5% phosphoric acid and destained with water…
Finding a good primary antibody can often feel like playing Russian roulette. Nothing is more disappointing than buying a $300 antibody that doesn’t work for your use. There are some steps you can take, however, to increase your likelihood of success. Scout out Other Labs Before you buy, ask if anyone around you or in…
2D gel electrophoresis (2DE) is a key technique for purifying individual proteins from complex samples based on their isoelectric points and molecular weights. Simple enough in theory, but as the plethora of protocols and articles shows, 2DE demands patience and meticulous optimization. But whether your samples are human sera or HUVEC lysates, 2DE uses these…
Transfection of animal cells has proven an invaluable tool in studies of gene expression, cell behavior, cell processes and molecular genetics. Essentially, transient pores are opened within the cell’s lipid bilayer, allowing insertion of nucleic acids (DNA, RNA, siRNA, RNAi), proteins, nanoparticles, and even antibodies into the cellular milieu. Transfection can be achieved using many…
Tandem affinity purification (TAP) is a versatile technique allowing the isolation of proteins for various purposes including Western blot and mass-spectrometry. The target protein is fused with protein A from streptococcus and the calmodulin binding domain, which together comprise the TAP-tag (for an introduction to TAP-tagging, see this article). To purify a TAP-tagged protein from…
LB medium is a staple in virtually every lab. It’s commonly used to propagate E. coli, and as such will be used frequently by any lab that does cloning. Chances are, LB broth or plates were one of the first things you learned to make as a newbie in the lab. Here are a few…
Co-immunoprecipitation is a method used to detect protein-protein interactions. While it can be wonderful when it works, there are many problems associated with this technique. One of the biggest problems that I have faced when using this method is contamination by the light and heavy chains of my precipitating antibody when performing western blots of…
Protein tags are invaluable tools for the modern biologist, particularly if you work on one of the 99% of proteins for which there isn’t a nice antibody readily available. If you want to purify large amounts of your protein of interest, detect it by western blot or fluorescence microscopy, or identify its potential binding partners,…
The last step in western blotting is imaging the blot – this is the moment of truth, when you finally get to see the results of the experiment you’ve been working on for so long! There are a variety of different ways to image your blot. The method you choose will largely depend on the…
The first step in most Western blotting experiments is lysing your cells to extract protein. You need to break open your cells in order to be able to isolate the proteins, and you need to do this with the least degradation and the most reproducibility possible. Depending on what your starting material is, there are…
Proteases: wild, mysterious, destructive. What are these untamed elements ravaging your precious lysate? How can a drop of EDTA or a smidge of “cocktail” protect that sample, which is gently cradling your hopes, your dreams, and your desire to survive the next lab meeting? Brace yourself for a biochem flashback: in this article, we’ll explain…
Western blots may be great for visualizing protein expression, but they can be a perfect way to waste your precious antibody stocks if you follow the normal protocol. Thankfully, you don’t have to follow the normal protocol any more; here’s how to get great blots with a fraction of the antibody usage. I have tried…
You’ve masterfully run and transferred your gel, and now it’s time to probe and quantify your protein(s). You’ve got your antibodies and ECL ready to go. Substrate – check. Film cartridge – check. Darkroom… Ah yes, that magical place where night vision goggles are required to navigate a veritable minefield of potential chaos. It didn’t…
When running a quantitative Western blot, it’s crucial that your sample preparation is consistent. Incomplete protein extraction from one sample will skew your results when you compare it to the protein content of a sample that was extracted more thoroughly. And after the protein extraction, it’s important to handle the samples in an identical manner…
When it comes to choosing a molecular weight marker to run on your SDS-PGE gels, there are a lot of options out there. How do you know which one is right for you? Read on for tips on what to consider when choosing a standard for your protein gels. Before you go about selecting a…
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…
I think that transferring Western blots is one the most enjoyable tasks to do in a lab: it’s quick, it’s messy, and on some gleeful level, it feels like a child’s art project gone wrong. Of course, it’s also finicky and slippery and prone to tiny pitfalls that can noticeably affect the quality of your…
For Western blot data to be reliable, it is important that you load known amounts of sample into each lane of the gel. This is of particular importance if you are doing a quantitative blot, where you really need to be able to compare band intensity in each sample. In this article, we’ll talk about…
Tandem affinity purification is a development of existing techniques for purifying protein complexes from cells in physiological conditions. It was first described over ten years ago and has become a commonplace laboratory tool. In this brief article I’ll introduce the basic technique and describe some of its advantages. Biology is a team game. Most biological…
RNA interference (RNAi) may have originated as a defense mechanism to protect cells against foreign genes introduced by viruses. This concept has since been put to use to create a powerful experimental tool for investigating gene function in organisms. Small-interfering RNA (siRNA) libraries for investigating genome-wide function can be produced by chemical synthesis of probes…
ELISAs (enzyme-linked immunosorbent assays) are often used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones in research and diagnostics. Today, a wide range of ELISA formats exist to suit your needs e.g. indirect ELISA, direct ELISA, competitive ELISA and sandwich ELISA. While it has become easier to perform ELISAs, thanks to…
Have you ever wished you could transfer the same SDS-PAGE gel twice? Sometimes, when you are blotting for many different proteins of similar size, stripping and reprobing multiple times can become impractical. Here’s a simple diffusion transfer method that can be used to generate duplicate membranes from a single gel: Protein transfer by this diffusion…
Phosphorylation is one of the major post-translational modifications that regulate the activity of a protein. Around a third of human proteins are believed to be phosphorylated, and so the kinases and phosphatases that mediate protein phosphorylation are of major interest to biomedical researchers. However detecting protein phosphorylation can be difficult, particularly from cell extracts. Phospho-specific…
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