Fluidic Analytics – Understand the Machinery of Life
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By building the world’s best tools, software and services for protein characterisation and making them universally accessible in the lab, in the clinic or at home, we are making this vision a reality not just for a small group of expert users, but for everyone who can benefit.
Do you need purified recombinant protein to test biological drugs? But you don’t have the time or facilities for cell culture and purification? Try using in vitro translation (IVT) instead!
IVT is like your own miniature protein factory. And it is great for a wide range of molecular biology applications because IVT can be much faster and simpler than traditional protein producing methods, (e.g. cell culture transfection and purification). IVT is done using ribosomes from any number of sources, including rabbit reticulocyte lysate, bacterial lysate, or even wheat germ extract. Nowadays, IVT is even available in kit form! Then all you need to supply is the recombinant mRNA, or DNA template.
Uses of In Vitro Translation
You could use IVT to produce control proteins for your Western blots, immunoprecipitations, ELISAs, or even to produce functional enzymes for affinity, kinetic, or thermodynamic experiments. But there are also many non-traditional uses for IVT. For example, in my lab, we use IVT to validate our protein fusion vectors. As an IVT experiment has a much faster turnaround than sending off a vector for sequencing, we use quick (2 hour) IVT assays to check that our DNA sequences are correctly inserted into our vectors and that our vectors produce full-length protein products. Just imagine all the things you could do with YOUR very own protein factory!
IVT Can Be Finicky Though…
With all the positives of IVT, there are some downsides to IVT. Primarily, IVT can easily have problems with producing appropriate protein yields or, even worse, it produces smaller protein fragments. These small fragments not only diminish the usefulness of your protein product but also can interfere with your assays—for example, a smaller fragment can give you incorrect kinetic measurements. Fortunately, you can take a few simple steps to ensure that you have good quality translation and excellent, high quality protein yields.
1. Use Clean Tools
First of all, take special care if you’re using an mRNA template that you produce yourself. However, the following applies whether your using your own mRNA template or coupling the in vitro transcription and translation with a combo kit. Your biggest risk is a damaged or degraded nucleic acid template. Outside of the friendly confines of a cell, a host of malicious cellular machinery easily degrades RNA.
The most notable malicious cellular machinery being RNAses—which are literally everywhere! RNases are on and in your own skin and sweat, where they act as a natural defense against microorganisms. From you, RNAses are easily carried off and spread around the lab as dust. So, however unfashionable, always wear your lab coat and gloves!
Also, to minimize RNase contamination, you must keep a clean lab bench. Therefore, before you begin any IVT assay, you need to clean your bench with 3% hydrogen peroxide or a broad-spectrum RNase decontamination solution, such as RNaseZap.
Additionally, I recommend that you purchase and use a dedicated set of RNase-free microcentrifuge tubes and pipette tips for all your RNA work (RNA synthesis, purification, or IVT). After all, dedicated pipettes are cheaper than redoing failed experiment after failed, contaminated experiment. And remember, whenever you are not using this dedicated equipment to keep it in a clean, secure place—away from other working areas of your bench and messy undergrads! I recommend placing all of your RNase-free equipment and consumables into sealed, plastic containers for storage to keep the dust out. Also, if you can afford it, consider investing in dedicated RNA only-centrifuges, vortexers, and other small equipment.
2. Use Fresh Ingredients
Another major source of RNase contamination is your solutions and buffers—even the water isn’t safe! The distilled or reverse osmosis-purified water that you normally use for your molecular biology work is not suitable for RNA work. Instead, make all of your solutions fresh using only RNase-free water, which is available from a number of companies. And remember: If you use ethanol or isopropanol to precipitate nucleic acids, you also need to dilute your alcohol stocks using RNase-free water!
Furthermore, to ensure that your new solutions are not contaminated by your glassware, bake your glass containers at 300°c for 2 hours (MMmmm I can smell it now) to destroy any persistent RNases that survived the washing process.
3. Use a Fresh, Clean Template
The third major issue with IVT is the production of truncated protein. These products often result from damage to your RNA/DNA template. Like the honey badger, ribosomal machinery “don’t give a d***” if your RNA template is intact. As long as the 5’ end of your template is there, the ribosomal machinery will bind and initiate translation. So, if you use truncated DNA/RNA templates, then you will produce truncated protein products.
Once made, truncated proteins are hard to remove from your reaction because they are chemically identical to your full-length products. So, your best bet is to not make them at all. And only you can prevent this by only producing and maintaining high-quality RNA/DNA templates.
For the best RNA/DNA templates:
- Avoid excessive pipetting and mixing.When you need to reconstitute your templates, do it gently by tapping and shaking with RNase-free water. Never use a vortex mixer to dissolve your template, as this can cause shearing (breaking) of your templates.
- Use fresh template.Whenever you can, use your templates immediately upon production. If you must stop before using them (because face it, sometimes you want to go home), then store your templates in ethanol at -80°c. And if you are going to use a single template for multiple IVT reactions, make numerous single-use aliquots to avoid multiple freeze-thaw cycles.