Compositions and methods for displaying antibodies in the periplasmic space of yeast cells are disclosed. In particular, antibodies are linked to a cell membrane-spanning transmembrane domain, a cell-membrane associated protein domain that is on the external face of the yeast cell membrane, a protein that binds to the inner face of the yeast cell wall, or a periplasmic protein in order to display the antibodies in the yeast periplasmic space. In addition, a target protein of interest can be coexpressed in yeast such that it is localized to the plasma membrane or periplasmic space and accessible to binding by displayed antibodies. The disclosure further relates to high-throughput screening of antibody libraries using yeast cell periplasmic display.

The present invention relates to chemical probes for the improved detection of cysteine in a test sample, preferably an aqueous test sample, as well as respective uses and kits.

Provided herein are methods of monitoring for the production of select antibodies in a non-human animal, comprising (a) immunizing a non-human animal with an immunogen; (b) obtaining a blood sample comprising antibody secreting cells (ASCs) from said non-human animal; and (c) individually assaying ASCs present in the blood sample, or a fraction thereof, for the production of select antibodies. Methods of guiding antibody production in a non-human animal for the production of select antibodies are also provided. In exemplary embodiments, the method comprises performing a cycle of (a) to (c), as above, and repeating the cycle when the percentage of ASCs producing select antibodies is below a threshold. In various aspects, the cycle is repeated until the percentage of ASCs producing select antibodies is at or above a threshold. Single cell assays are further provided herein.

A light-inducible intracellular protein aggregation system is described herein, which provides invaluable tools to study the role of protein aggregates in proteinopathies and to screen for novel therapeutic compounds. The system generally comprises a cell expressing an alpha-synuclein polypeptide or another proteopathic polypeptide that self-aggregates under pathogenic conditions, operably linked to a photoactivatable polypeptide. Illumination of the photoactivatable polypeptide with light having a wavelength sufficient for photoactivation triggers irreversible accumulation of intracellular protein aggregates comprising the alpha-synuclein polypeptide or proteopathic polypeptide. The intracellular protein aggregates can be made to accumulate in real-time during the illumination, thereby enabling spatiotemporal control of protein aggregation. In some embodiments, the intracellular protein aggregates may exhibit pathologically-relevant properties of those found in disease-associated proteinopathies, such as irreversibility, auto-perpetuation (seeding) activity, and comprising misfolded proteins rich in beta-sheet conformation.

A method of determining the effect of non-specific interactions in simulated in vivo conditions is presently disclosed. The method includes (a) contacting a solution comprising a biologically relevant molecular crowding agent and a target molecule with a biosensor, wherein the surface of the biosensor comprises a capture molecule that specifically binds the target molecule; (b) allowing the target molecule to bind to the capture molecule; and (c) determining an amount of the target molecule bound to capture molecule using biolayer interferometry.

Provided herein are methods of generating hybridomas and related methods of producing antigen-specific antibodies. In exemplary embodiments, the method comprises (a) preparing an enriched population of IgG-positive (IgG+) memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein (i) less than or about 10% of the enriched population are IgM-positive (IgM+) B cells and/or (ii) the ratio of the IgG+ memory B cell count to IgM+ B cell count of the enriched population is greater than about 0.5, optionally, greater than about 1 or greater than about 2, (b) bulk-culturing the enriched population to obtain an expanded population; and (c) fusing cells of the expanded population with myeloma cells to obtain hybridomas. In exemplary aspects, the hybridomas obtained represent at least 10% or at least 15% of the IgG+ memory B cell repertoire produced by the immunized animals.

The present invention relates to a polypeptide comprising an antigen binding domain and a carrying moiety having an inhibiting domain that inhibits the antigen binding activity of the antigen binding domain, and having a longer half-life than that of the antigen binding domain existing alone, methods for producing and screening for the polypeptide, a pharmaceutical composition comprising the polypeptide, methods for producing and screening for a single-domain antibody whose antigen binding activity is inhibited by associating with particular VL, VH or VHH, and a fusion polypeptide library including a single-domain antibody whose antigen binding activity is inhibited by associating with particular VL, VH or VHH.

The present invention relates to G protein peptidomimetics capable of stabilizing a GPCR in an active conformational state. The G protein peptidomimetics are derived from the G protein epitope interacting with the GPCR, in particular from the C-terminus of the as helix comprising the amino acid sequence FNDCRDIIQRMHLRQYELL (SEQ ID NO:117). The invention further provides complexes of the G protein peptidomimetics and a GPCR, fusion polypeptides of a GPCR and the G protein peptidomimetics and compositions comprising the same. Further disclosed herein are uses of the G protein peptidomimetics, complexes, fusion polypeptides and compositions for determining the structure of the GPCR conformer and for screening for compounds capable of specifically binding to the GPCR conformer.

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

Provided herein are various methods for identifying candidate substrates for the E3 ligase machinery.