Biochemistry

Peptergent is a novel class of amphipathic peptides that enables detergent-free extraction of membrane proteins (MPs) from lipid bilayers. This reagent self-assembles around hydrophobic transmembrane regions, forming stable, water-soluble complexes that can be isolated directly from biological membranes. Peptergent therefore bypasses the limitations imposed by traditional detergents, which often destabilize protein assemblies. Since detergents are completely avoided, MPs are directly amenable to structural and mass spectrometry (MS) analysis, thereby addressing their persistent underrepresentation in proteomic datasets and improving their accessibility in drug-screening strategies. We present here a streamlined protocol for MPs extraction with the Peptergent PDET-1, followed by exchange into His-tagged Peptidiscs for Ni-NTA-based affinity purification. The method encompasses membrane isolation, peptide preparation, protein extraction, clarification, and MPs exchange from Peptergents to Peptidiscs. This workflow yields an enriched membrane proteome compatible with downstream LC-MS/MS analysis for improved identification of multi-pass MPs.

Cellular function relies on a network of precisely regulated interactions among macromolecules such as proteins, peptides, carbohydrates, and nucleic acids. These molecular interactions regulate vital processes, including signaling, structural organization, and developmental patterning. Biolayer interferometry (BLI) is a label-free optical biosensing technique that enables real-time quantification of such interactions. This protocol describes how to use BLI to assess the binding affinity between a biotinylated plant peptide hormone (RALF1) and cell wall–derived oligogalacturonides (OG25–50) on the Octet RED96 platform. Streptavidin-coated biosensors are employed to immobilize the ligand, while analyte binding is monitored through wavelength shifts in the reflected light. The protocol includes detailed steps for sensor preparation, assay setup, software configuration, and kinetic data analysis. While optimized for plant peptide–matrix interactions, the method is broadly adaptable to other macromolecular systems across biological disciplines.

The genome of the nematode Caenorhabditis elegans encodes at least 160 predicted peptide precursor genes that can generate over 300 bioactive peptides, the functions of most of which remain unknown. Phenotypes resulting from deletion or transgenic expression of peptide genes are readily assayed, but genetic dissection of individual peptide activities is often confounded when a single gene encodes multiple peptides or when distinct peptides act redundantly. Here, we describe a protocol for direct microinjection of chemically synthesized peptides into individual worms. This approach permits investigation of the effects of an individual peptide while providing precise temporal control over peptide delivery.

When performing renal biopsy, it is necessary to identify the cortex, where glomeruli are exclusively distributed, to ensure the quality of the specimen for histological diagnosis. However, conventional methods using a stereomicroscope or magnifying lens often fail to clarify the quality of the specimen. We have established a fluorescent-based imaging technique for the on-site assessment of renal biopsy specimens. The fluorescent images can be easily obtained by adding an optical filter to the microscope and with a short incubation of an activatable fluorescent probe. This novel imaging technique can be applied to renal biopsy specimens for distinguishing the renal cortex.