Stability & formulation screening
The problem
Misbehaving proteins can unfold, aggregate and ruin everything, but the right protein construct will hold it together and save the day. Once you’ve got the right protein, picking formulations is the next big hurdle. Screening for the best proteins and formulations requires having all the right data to advance the best candidates.
Outdated ways of looking at protein stability take way too much sample and time, and often deliver only part of the story. To keep an eye on all the ways that protein stability can go wrong, unfolding and aggregation have to be measured at the same time on the same sample. Plus, the earlier you can weed out bad candidates, the better – so it’s critical to use tiny amounts of protein.
The right tool for the job
Uncle is the all-in-one formulation screening and stability platform that enables more than twelve different applications to quickly assay protein stability - on only 9 µL of sample. Fluorescence, static light scattering (SLS) and dynamic light scattering (DLS) modes all read the same sample and combine to give powerful insights such as thermal melting, aggregation, and sizing in a ramp experiment or during constant temperature incubation.
To get the whole story from your protein stability assay, you need to combine SLS and fluorescence. While fluorescence sees unfolding, SLS will spot aggregation the instant protein molecular weight changes. Run up to 48 samples at a time and get results in less than 2 hours so you can screen more candidates, formulations, or conditions to quickly figure out which is the most stable.
The proof
How Uncle spots stability problems
A DSF experiment on Uncle starts with using DLS to look at size and size distribution right off the bat, so you know if your protein is good to go or if it’s already suffering from aggregation. Next, as Uncle heats up your samples, fluorescence picks up when protein unfolding occurs and calls out any melting temperatures (Tm). SLS then shows when aggregation begins as an aggregation temperature (Tagg) – and that can sync up with Tm1, Tm2, or happen independently from any Tm. Because SLS intensity is related to molecular weight, it's incredibly sensitive to aggregation and is the best way to see exactly when aggregation begins. After the thermal ramp maxes out temp, DLS can be used once more to quantify how bad aggregation has gotten.
Accelerate stability testing
Thermal ramps characterize and rank your samples in about two hours, but a full, detailed look at stability also needs longer incubations at constant, elevated temperatures. Uncle opens the door to getting isothermal data earlier in candidate selection with a suite of apps tailor-made to monitor protein behavior over hours or days right inside Uncle. You can also rack up long term data while keeping your Uncle free for other experiments with its out-of-the-box isothermal applications – load your samples into Uncle cuvettes, stow those in an incubator, and then pop them back into Uncle to read data once a day or once a week – it’s your call.
Uncle’s isothermal applications let you focus in on protein fluorescence or the signal from a reporter dye like SYPRO Orange – so it’s no problem to study non-fluorescent proteins or formulations that quench fluorescence. SLS and DLS chart aggregation throughout isothermal testing, keeping you on top of all stability parameters all the time. With its one-of-a-kind isothermal apps, Uncle is your stability platform for an early, low volume look at accelerated, isothermal storage testing.
Mix it up
Uncle is all about formulation screening and allows you to compare proteins, formulations, and everything else that impacts stability. A simple amino acid mutation or change of buffer can increase or decrease melting temperatures or can change unfolding behavior so that a protein with two melting temperatures now has three. Similar kinds of changes can happen with sugars, salts, surfactants, pH shifts or other protein hazards like freeze/thaw cycles.
Stack the deck
Beyond the classic DSF or isothermal experiments as protein stability assays, the flexibility of three detection technologies teamed up with temperature control makes Uncle a jam-packed toolbox of protein stability. Unfolding and aggregation data is just the gateway to isothermal stability, protein self-interaction, refolding, viscosity, and more. Uncle has over a dozen applications designed to rapidly extract the most possible data from the smallest sample volume of protein.
Uncle
Cracking stability using a pile of one-trick, protein-hungry tools is a ton of work. Uncle combines 3 different measurement modes — fluorescence, Static Light Scattering (SLS) and Dynamic Light Scattering (DLS). So you can crank out all your data in just a few hours, and use way less protein. All the info you’ll get makes picking the best formulation, protein, or viral vector a piece of cake.
Want more info?
Want to learn more about how Uncle makes it easy to screen for the most stable protein or best buffer?