生物化学

Nowadays, recombinant proteins are the focus of various research fields, and their use ranges from therapeutic investigations to cellular model systems for the development of therapeutic approaches. Cell systems used for the expression of recombinant proteins should be comparable in terms of yield and expression efficiency. In many research fields, it is desirable to obtain high protein concentrations. A method that combines an easy workflow with rapid results and affordable costs remains missing, and a standardized approach to determining protein concentration in transgenic cell lines is essential for more reliable data analysis. Our protocol demonstrates the cluster fluorescence-linked immunosorbent assay (FLISA), a technique that allows the exact quantification of comparable protein expression amounts. Moreover, it enables the detection of clustered or bound subunits of a protein without necessitating ultracentrifugation. In the present protocol, we demonstrate the utilization of two transgene cell lines, each expressing distinct recombinant proteins, to provide comparability of protein yields and detectable subunit clustering.

Cellular phenomena such as signal integration and transmission are based on the correct spatiotemporal organization of biomolecules within the cell. Therefore, the targeted manipulation of such processes requires tools that can precisely induce the localizations and interactions of the key players relevant to these processes with high temporal resolution. Chemically induced dimerization (CID) techniques offer a powerful means to manipulate protein function with high temporal resolution and subcellular specificity, enabling direct control over cellular behavior. Here, we present the detailed synthesis and application of dual SLIPT and dual SLIPT^NVOC, which expand the SLIPT (self-localizing ligand-induced protein translocation) platform by incorporating a dual-ligand CID system. Dual SLIPT and dual SLIPT^NVOC independently sort into the inner leaflet of the plasma membrane via a lipid-like anchoring motif, where they present the two headgroup moieties trimethoprim (TMP) and HaloTag ligand (HTL), which recruit and dimerize any two ^iK6eDHFR- and HOB-tagged proteins of interest (POIs). Dual-SLIPT^NVOC furthermore enables this protein dimerization of POIs at the inner leaflet of the plasma membrane in a pre-determined order and light-controlled manner. In this protocol, we detail the synthetic strategy to access dual SLIPT and dual SLIPT^NVOC, while also providing the underlying rationale for key design and synthetic decisions, with the aim of offering a streamlined, accessible, and broadly implementable methodology. In addition to the detailed synthesis, we present representative applications and typical experimental outcomes and recommend strategies for data analysis to support effective use of the system. Notably, dual SLIPT and dual SLIPT^NVOC represent the first CID systems to emulate endogenous lipidation-driven membrane targeting, while retaining hallmark advantages of CID technology—the precision over POI identity, recruitment sequence, high spatiotemporal control, and “plug-and-play” flexibility.

In the field of osteoarthritis (OA), the identification of reliable diagnostic and prognostic biomarkers in patients with hip lesions such as femoroacetabular impingement (FAI) could have an immeasurable value. Calcium crystal detection in synovial fluids (SFs) is one tool currently available to diagnose patients with rheumatologic disorders. Crystals, such as monosodium urate (MSU) and calcium pyrophosphate (CPP), are identified qualitatively by compensated polarized light, whereas basic calcium phosphate (BCP) crystals are visualized under conventional light microscopy by Alizarin red S (ARS) staining. Here, we present an efficient and straightforward protocol to quantify calcium crystals by spectrophotometric analysis in human osteoarthritic SFs after staining with ARS. The type and size of the different crystal species are confirmed by environmental scanning electron microscopy (ESEM).

Liquid–liquid phase separation (LLPS) underlies the spatial organization of the nucleolus, a membraneless organelle responsible for ribosomal RNA (rRNA) transcription and ribosome subunit assembly. One of the key proteins involved in the formation of the fibrillar center of the nucleolus is the treacle, an intrinsically disordered protein that contains low-complexity repeats enriched in charged amino acid residues. In this work, we present a detailed protocol for the bacterial expression and purification of a recombinant fragment of treacle comprising two tandem low-complexity repeat (LCR) modules, with a total length of 136 amino acids. This fragment is intended for subsequent in vitro investigation of its ability to undergo LLPS. The described method enables the production of a soluble, biochemically pure protein preparation suitable for studying the mechanisms of spontaneous condensate formation in a cell-free system. This approach allows for the controlled modeling and quantitative evaluation of the contribution of low-complexity sequences to the phase behavior of treacle, independently of its interactions with cellular partners in vivo.

N-glycosylation is a ubiquitous post-translational modification (PTM) that regulates protein folding, stability, and biological function. Accurate identification and validation of N-glycosylation are therefore critical for understanding how glycosylation modulates protein activity. Here, we present a robust workflow for analyzing protein N-glycosylation in both animal and plant systems using peptide-N⁴-(N-acetyl-β-glucosaminyl) asparagine-amidase A and F (PNGase A and PNGase F). After enzymatic cleavage of the asparagine-linked N-glycans, samples are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB) to detect shifts in apparent molecular weight (MW) indicative of deglycosylation. Key steps include denaturing the protein to expose glycosylation sites, optimizing buffer conditions for PNGase F and A treatment, and comparing glycosylated vs. deglycosylated forms by electrophoretic mobility. A troubleshooting guide addresses common challenges, including incomplete deglycosylation and low transfer efficiency during WB, offering practical solutions to ensure reliable results. This protocol provides researchers with a standardized, cost-effective framework for investigating protein N-glycosylation in diverse systems, from cell lysates to purified proteins, in both animal and plant models.

Candida albicans is the pathogenic fungus that most frequently causes infections in humans. It is part of the microbiota commonly found in the skin, gastrointestinal tract, and vaginal mucosa. However, certain conditions, including immunosuppression, excessive use of antibiotics, hormonal changes, the use of medical devices in patients, and individual nutritional status, promote the development of opportunistic infections caused by this fungus. One of the main fungal structures interacting with the host is the cell wall, which is principally composed of chitin, glucan, and proteins. The cell wall plays key functions for the cell, such as osmotic protection; it is also responsible for cellular shape and acts as a signaling hub in response to environmental changes. Cell wall proteins participate in diverse cellular functions, such as attachment to surfaces and cell wall structure; some possess catalytic or transport activities. In this protocol, we show the methodology for isolating cell wall proteins covalently linked or not to cell wall components that can be previously labeled with [¹⁴C]-L-lysine by the action of the fungal transglutaminase localized in the cell wall. We use an extraction method by mechanical cell disruption and washing with 2 M NaCl, whose ionic strength eliminates contaminating proteins from other organelles, through subsequent serial treatments with SDS, chitinase, and zymolyase.

This protocol describes a standardized and economically accessible method for synthesizing mRNA-encapsulated lipid nanoparticles using routine laboratory equipment, including precision syringe pumps, Y-shaped glass microfluidic chips, and silicone tubing. Designed to address the cost and accessibility limitations of commercial microfluidic platforms, the system achieves performance metrics comparable to high-end devices while reducing equipment costs by 90%. By systematically optimizing hydrodynamic parameters (total flow rate: 12 mL/min; lipid-to-aqueous phase ratio: 3:1), the protocol enables consistent production of lipid nanoparticles with key quality attributes: high mRNA encapsulation efficiency (≥ 80%), narrow particle size distribution (100–120 nm, polydispersity index ≤ 0.2), and excellent storage performance (≥ 7 days at 4 °C ).

OtUBD is a high-affinity ubiquitin-binding domain (UBD) derived from a large protein produced by the microorganism Orientia tsutsugamushi. The following protocol describes a step-by-step process for the enrichment of ubiquitinated proteins from baker's yeast and mammalian cell lysates using OtUBD. The OtUBD affinity resin can strongly enrich both mono- and poly-ubiquitinated proteins from crude lysates. The protocol further describes the use of different buffer formulations to specifically enrich for proteins covalently modified by ubiquitin with or without proteins that associate with them. Combining different OtUBD-mediated enrichment protocols with liquid chromatography–tandem mass spectrometry (LC–MS/MS) helps distinguish the pool of covalently ubiquitinated proteins (the ubiquitinome) from ubiquitin- or ubiquitinated protein-interacting proteins (the ubiquitin interactome). The OtUBD tool described in the protocol has been used successfully with downstream applications such as immunoblotting and differential proteomics. It provides researchers with a versatile and economical tool for the study of ubiquitin biology.

The phototransduction cascade allows photoreceptors to detect light across a wide range of intensities without saturation, with cGMP serving as the second messenger and calcium feedback as the key regulatory mechanism. While experimental evidence suggests that cAMP may also play a role in modulating this cascade, such regulation would necessitate rapid changes in cAMP levels on a timescale of seconds. However, data on the dynamics of intracellular cAMP changes in photoreceptors remain scarce, primarily due to the limitations of conventional fluorescence-based methods in this specialized sensory system. To address this gap, we developed a methodology combining rapid cryofixation of retinal samples following light stimulation with the isolation of outer segment preparations. The rapid cryofixation setup comprises six computer-controlled sections, each with a high-speed stepper motor-driven lever that rapidly moves the specimen in a 180° arc within ~80 ms to press it against a liquid nitrogen-cooled copper cylinder for fixation. Using highly sensitive metabolomics techniques, we measured cAMP levels in these samples. This approach enables the investigation of rapid cAMP dynamics and its potential regulatory role in phototransduction, providing a foundation for understanding the interplay between cAMP and PKA signaling in photoreceptor function.

Rhamnogalacturonan-II (RG-II) is one of the least studied domains of pectin, primarily due to its low abundance, the lack of reliable antibodies, and the complexity of its structure. The present study builds upon existing protocols and procedures used to analyse RG-II in tissues where it is more abundant, combining and adapting them for the isolation of RG-II from Arabidopsis seed mucilage—a structure previously thought to lack RG-II. By applying these adapted methods, we first confirmed the presence of RG-II in seed mucilage and subsequently succeeded in isolating it from a tissue where it is typically present in low abundance, thereby enabling future studies on this previously overlooked component.