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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.
Has your supervisor asked you to do a polysome profile? Are you attempting them but running in to problems? This article will help explain what they are, the basics of the protocol and provide some helpful tips (including some things that I wish I had known when I first started out). Polysome Profiling: The Basics…
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…
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….
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.
Struggling with your PhD advisor relationship? Sometimes its repairable and sometimes it’s better to switch. Discover how to identify when it’s time to switch advisors and steps you can take to ensure a smooth successful change to keep your research and academic goals stay on track.
One of the most exciting things about being a scientist is doing experiments that have never been done before. Unfortunately, this can also be one of the most daunting aspects of science. Read on to make taking on your next experiment less daunting.
If you do cell culture you will inevitably need to count your cells. Counting cells can be tedious, but it is important to do accurately. Your assessed quantity of living cells will affect all your downstream applications. In this article I will not only cover how to manually count your cells and how to do…
Like most other chromatographic techniques, thin layer chromatography (TLC) separates out individual compounds from a mixture depending upon the polarity of each compound. The solvent system travels up a silica plate by capillary action and passes over the sample that you spot onto the plate. As the solvent travels up, it moves the compounds present…
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…
After you’ve generated your mutuations using CRISPR-Cas, the next step is to identify those cells that have been successfully edited. There are a few different ways to check for the mutations. I’m going to discuss some of the more popular ones.
Quantitative PCR (qPCR) uses fluorescent dyes or probes to visualize the amplification of specific DNA sequences as it happens (i.e. in real time). The dyes or probes fluoresce when they bind to newly amplified DNA, and the amount of fluorescence emitted is proportional to the amount of DNA (or mRNA) present in the original sample. By detecting newly synthesized DNA…
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.
In the sci-fi novel Terminal World by Alistair Reynolds, a planet consists of zones with defined characteristics of matter interactions on a subatomic level. These conditions permit different levels of technology sophistication in various zones. For example, in the “Steamville zone” nothing more complicated than steam engines works – electronic schemes fuse irreversibly. Something like…
Retroviral transduction is becoming a popular choice for gene delivery into mammalian cells and has multiple advantages over other techniques. If you decide to start work on this useful technique, here is how you can go about it: Step 0: Obtain permission First and foremost, do you have the permission, authorization, and training to work…
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.
Alu sequences are repetitive DNA sequences that are widely dispersed within the human genome. These “junk DNAs” are not as useless as one might think. An interesting method to use them is to quantify the number of integrated Human Immunodeficiency Virus (HIV) genome copies using Alu-PCR.
The ability for DNA polymerase to copy a long stretch of DNA is becoming increasingly important. Why? It has to do with the advances in our sequencing technologies. Our next generation sequencing (NGS) technology requires the DNA polymerase to copy a long stretch of DNA (sometimes up to 50kb) as NGS is churning out genetic…
Generating a DNA vector library of single and/or compound mutants for a target protein can be a daunting task. If you’re lucky and work in a well-funded lab, you might outsource this process via gene synthesis. Most of us though, need to do it the old-fashioned way. Traditional QuikChange™ Traditionally, there are many steps you…
Say you just joined a lab and have been assigned your very own project to work on. As part of your new responsibilities, you have to breed and maintain the mutant (or transgenic) mouse line which you will be using for your experiments. An integral part of mouse genetics experiments is determining the genotype of…
Greetings, I’m a research student and I’m having a lot of trouble in finding the right antibodies for my study, because I need both monoclonal and polyclonal. And I need to start addressing some other international scientists. The problem is that I don’t know how to address the situation. If they indeed have antibodies that…
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…
Do you know about algae and their potential in today’s world? Do you know how to work with algae? Algae are becoming increasingly important in the research world.
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….
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…
Lab outreach programs are one of the ways that science reaches out to its future generations and entices them to join the ranks of those who have pledged allegiance to the pursuit of the unknown. Successfully running these programs for young adolescents depends on a certain number of factors, such as the ability to capture…
Pat yourself on the back, you saw a post with the word “coding” in the title and you didn’t freak out or glaze over. That’s the first step. Coding seems to have such a stigma attached to it; people tend to think that it’s incomprehensible nonsense that they could never learn, and that it has…
While it is true that there are some useful websites like SNPedia, or NCBI that can help you find rs codes for genetic variants, sometimes you need that info coming straight from the oven – particularly when you want to look at atypic SNPs or substitutions that have not been validated. So, in this post I…
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!
Lab work, as we are all aware, comes with many pressures: one of which is productivity. You want to generate as much quality data as possible to meet publication deadlines or perhaps the elusive thesis. Sometimes it may feel like hours spent in the lab don’t match the amount of data produced: for some this…
Here are some ideas to make your liquid handling robot perform at its best.
When I began a master’s program in 2008, the lab task I hated more than anything was running agarose gels for DNA. Something so simple, ubiquitous, and necessary gobbled up more hours in the lab than I care to remember. Even though we added the DNA stain directly to the molten agarose and didn’t have…
After ten years of postdoctoral research there is one important piece of advice I would give to anyone embarking on a research career: Spend as much time managing your data as you do generating it Take time at the beginning of each project to organize how you will record what you are doing day-to-day. The…
Welcome to the magical world of systematics! Looking for a way to produce a phylogenetic tree that’s a step above the default options, time efficient, not too program heavy and avoids using command line programs? Although there are more rigorous analyses that strict systematists perform, for your purposes, the following should suffice. 1. Data selection…
The gap year I intended to take between my Master’s degree and hypothetical Ph.D. is now going into its 4th year. Here’s why I’m not worried. These days it seems like undergraduates are proceeding en masse to graduate programs shortly after completing their senior year of college. An abundance of undergraduate research opportunities and poor…
Three dimensional cell culture mimics the extracellular matrix (ECM) that offers the structure and support for cells in vivo, thus creating the complex architecture and network required for cellular communication. For 3D cell culture beginners (or enthusiasts), the information available may seem overwhelming. It sure was for me. But it can be simplified. For example,…
The more experienced hands in your lab know that molecular biology is rarely just a journey from A to B. As a result, I’ve constructed this short workflow as an introduction to genomic molecular techniques.
Producing lentiviral or retroviral vectors is theoretically fairly straightforward. However, anyone new to viral vector work is usually confronted with vast amounts of confusing information. It seems that anyone who has ever made a lentivirus has their own protocols and is adamant that their method is the best one to follow. In reality, there are…
You can’t put it off any longer – you need to write your first manuscript. This task can seem daunting at first, but with a little organization and planning, you’ll soon be publishing.
Some tissues are tricky to work with. This truth was lost on me in the early years of grad school because I worked with liver samples. If you’re extracting RNA from liver samples, you’re likely not losing sleep over your massive RNA yields. But for the folks doing RNA extractions with less willing donors, such…
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