 Hi, I'm Mark Fisher, and today Dr. Kunu and I are going to discuss or outline some of the results here in this month's protein science issue entitled Proving structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to biolayer interferometry In this publication, we demonstrate that we can use the chaperone-based biolayer interferometry biosensor system, or the GroEL biosensor, to capture and detect the appearance of pre-aggregated species or what we call the seeds of destruction in protein solutions before large scale aggregation sets in. So the GroEL biosensor binds these partially folded proteins that have exposed hydrophobic surfaces and this binding is easily reversed using ATP or ATP osmolite solutions and this reversibility in binding is actually a hallmark of the specificity in this specific binding with this technique. So Dr. Kunu is actually going to discuss some of the highlights of this work. Biolayer interferometry or BLI is a label-free method that can measure changes in protein interactions through changes on a fiber optic biosensor tip by monitoring changes in the interference patterns. We use the single-channel BLITS system for our measurements. The system sets up an interference pattern based on reflectance from a reference layer that is compared with the biolayer establishing constructive and destructive interference patterns. As the sample thickness on the tip changes, resulting in a kinetic and amplitude change, results in a phase shift that yields a real-time kinetic BLI sensor. As just one example, we use the system to detect the existence of pre-aggregates in polyclonal, monoclonal, and antibody solutions. We optimize the GroEL binding concentrations to eliminate nonspecific binding. An increase in signal occurs if partially-folded proteins become bound to the GroEL biosensor. Alternatively, when the solution contains ligand or solution-stabilizing conditions that do not bind or interfere with GroEL, this binding signal can diminish or, in cases where tight binding protein stabilizers are present, the signal can completely disappear. As just one of the many examples illustrated in this paper, as one applies moderate heat to monoclonal antibody solutions, there is a contaminant rise in signal of protein binding to GroEL. This binding is specific since ATP addition to GroEL reverses the binding, and the BLI signal returns to baseline. We also demonstrate that we can use a reversible cleavable disulfide biotin linkage to the streptabbitin tip, where one can release GroEL antibody complexes and visualize these complexes using negative stain electron microscopy, using as little as three microliters. In this again image, one can see that protein components have become bound to the GroEL from the BLI tip, and in some cases, an entire antibody molecule is present, either as bound or dissociated from the GroEL platform. So in summary, we've been able to detect pre-aggregates of three separate protein solutions, polyclonal antibody, monoclonal antibody, and the fiberglass growth factor prior to large-scale aggregation. We plan to use this GroEL BLI biosensor platform to determine if early pre-aggregate detection is linked to longer-term aggregation. This also should allow us to identify and validate ligand or solution protein stabilizers, and to test out actually engineered stabilities in protein structure. And finally, our ability to visualize intermediates bound to GroEL using electron microscopy may enable us to identify regions that are particularly within antibodies that are susceptible to local unfolding using a technique called EM single particle analysis. So with that, thank you for listening and stay tuned.