BioTechniques最新文献

This protocol describes a methodology combining TurboID - a recently developed proximity biotinylation technique - with conventional epitope-tag based co-immunoprecipitation (Co-IP) to analyze protein-protein interactions (PPIs) in cell culture systems. This integrated approach allows the targeted examination of both transient and stable protein interactors, enhancing our understanding of protein dynamics. TurboID captures transient interactions often missed by Co-IP, which captures high affinity, stable interactors. Combination of both techniques enables direct comparison of interaction strengths, providing insights into the dynamic nature of protein interactions within cells. The rapid biotinylation capability of TurboID reduces background noise and false positives while Co-IP enriches stable interactors, together improving data quality and interpretation of the interactomic landscape. Demonstrating the efficacy of this methodology, proteins relevant to the pathology of Spinal Muscular Atrophy were utilized to explore variations at the interactome level. The use of identical starting cell lysates for both TurboID and Co-IP minimized variability and ensured datasets were comparable, allowing for consistency and enhancing the reliability of findings regarding the nature and strength of protein interactions. This novel framework effectively combines both innovative and classical techniques while maintaining consistency in sample handling, advancing our understanding of the intricate networks that govern cellular processes.

Full data-set available at https://www.ebi.ac.uk/pride/ reference ID PXD066858.

Phage display combined with biopanning is a powerful method for the discovery of therapeutic antibodies. Classic biopanning is lengthy and labor-intensive, requiring at least five days of work. The biopanning process is plagued by a bias toward binders with propagation advantages, causing a loss in diversity. We present a more efficient biopanning technique named 3in1 biopanning. Using non-denaturing elution and skipping the intermediary phage amplification steps, 3in1 biopanning performs all biopanning rounds in a single day and overcomes the inherent biopanning bias. 3in1 is five times faster than conventional biopanning and yields up to six times more unique hits with a wide range of affinities. It shortens lead discovery time by a week and reduces consumables and waste. We demonstrate that this novel biopanning technique is suitable for naive-synthetic and immune libraries against a variety of targets.

Bovine serum albumin (BSA) exhibits lot-to-lot variability, partly due to differences in fatty acid content. In this study, the structural properties of two BSA lots-fatty acid-bound and -free-were compared using electrophoretic, chromatographic, and spectroscopic techniques. No apparent differences in overall structure were detected by conventional methods, including UV absorbance spectroscopy, reducing and non-reducing SDS-PAGE, gel filtration chromatography, or agarose native gel electrophoresis. However, circular dichroism (CD) analysis of native BSA revealed small but significant differences in folded structure between the two lots, particularly in the microenvironment surrounding one of two tryptophan residues, a known fatty acid-binding region. These differences were further supported by the intrinsic fluorescence measurements. Under heat stress (73-76 °C), the two lots exhibited distinct behaviors. Native gel electrophoresis and CD spectroscopy conformational states with different patterns, including variations in aggregation propensity. These results demonstrate that, for the first time, combining CD and fluorescence spectroscopy with native electrophoresis under heat-stress conditions provides a sensitive and practical approach for detecting subtle conformational differences among BSA lots, offering a valuable tool for assessing protein quality and consistency.

The presence of 5-methylcytosine at CpG sites in mammalian DNA plays a significant role in various biological processes, both normal and aberrant. Similarly, CpG site methylation status of therapeutic DNAs can affect activity and therapeutic efficacy, as is the case for ADI-100, an immune tolerance-inducing drug candidate for the treatment of type 1 diabetes and other GAD-associated autoimmune diseases. To assess the methylation characteristics of ADI-100 and other therapeutic DNA product candidates, we developed a simple and versatile method, methylation-sensitive restriction enzyme digest and agarose gel electrophoresis (MSRE-AGE), using methylation-sensitive restriction enzyme digest, agarose gel electrophoresis, and custom in silico band pattern analysis for quantitative measurement of CpG methylation levels and patterns. We compared MSRE-AGE with bisulfite pyrosequencing methylation analysis of plasmid DNA and a synthetically-produced closed-linear DNA, Doggybone DNA (dbDNA), demonstrating that MSRE-AGE produces results consistent with bisulfite pyrosequencing but without structure and sequence-dependent artifacts. These findings demonstrate that MSRE-AGE is a robust and practical method for methylation analysis of therapeutic DNA products.

Plasmid DNA remains the standard tool for mammalian transfection but carries bacterial backbone sequences that can reduce expression efficiency and raise biosafety concerns. Minicircle DNA eliminates these elements and improves expression but requires bacterial recombination systems and multi-day protocols. Here we present Self-circularized Overhang-based Unrecombined Products (SOUP), a rapid in vitro workflow for generating backbone-free circular expression cassettes directly from polymerase chain reaction (PCR) products. The method involves cassette amplification with engineered overhangs, type IIS restriction digestion, self-ligation, and RecBCD exonuclease treatment, producing circular monomers alongside dimers and concatemers in less than 24 hours. We evaluated SOUP against its parental plasmid carrying an identical green fluorescent protein (GFP) cassette in Human embryonic kidney 293 (HEK293) cells. At 24 h post-transfection, SOUP yielded a larger proportion of GFP-positive cells, while the plasmid supported higher per-cell intensity. By 56 h, SOUP showed a clear advantage, with increased mean fluorescence, higher transfection efficiency, and a productivity index more than 2.5-fold greater than the plasmid. These results demonstrate that SOUP constructs, despite their heterogeneous composition, support strong gene expression. The workflow is fast and inexpensive, making it accessible as a practical complement to plasmid vectors and a rapid prototyping tool for backbone-free DNA constructs.

Introduction: Low-density lipoprotein receptor-related protein 8 (LRP8) is a neuronal receptor for apolipoprotein E and Reelin, two ligands critically involved in Alzheimer's disease (AD), neuronal migration, and memory. Because LRP8 is highly expressed in neurons, interacts with amyloid precursor protein, and undergoes γ-secretase-dependent processing, it has emerged as a potential contributor to AD-related neurodegeneration. Growing evidence also implicates LRP8 in carcinogenesis, highlighting the need to better define its molecular properties. Areas covered: This article addresses the limited understanding of LRP8 proteolytic processing, cellular localization, and molecular interactions, due in part to the lack of suitable antibodies. We present and characterize novel polyclonal and monoclonal antibodies directed against the C-terminal region of LRP8, suitable for Western blotting and immunocytochemistry/immunofluorescence. These reagents enabled detection of a previously unrecognized intracellular low-molecular-weight (∼12 kDa) C-terminal LRP8 fragment. Expert opinion/Commentary: These antibodies provide valuable new tools for mechanistic studies of LRP8. By improving the investigation of LRP8 processing and localization, they may facilitate a better understanding of its role in neurodegeneration and cancer.

Previous molecular methods for sex determination of sturgeon of the Acipenser genus have been repeatedly validated for females, but have largely relied on suboptimal solutions to compensate for the lack of a confirmed control for males. By combining sturgeon cytochrome b (cyt b) gene amplification as a reference within existing qPCR techniques, this limitation is addressed through the demonstration of both the presence and confirmed absence of the female-specific AllWSex2 amplicon. Duplexing produces distinct melting curve profiles that enable simultaneous evaluation of both Acipenser confirmation and female identification targets. Incorporating the cyt b primer pair results in clearly distinguishable peak patterns for reliable sex determination, eliminating the need for additional probe detection, and making the approach more cost-effective, faster, and efficient than traditional multiplexing techniques. The method also highlights the importance of developing and appending existing assays to include said endogenous reference controls, such as the cyt b marker used here.

This study evaluated the reduction of selected antibiotic resistance genes (ARGs) in a wastewater treatment plant (WWTP) using conventional activated sludge and assessed the distribution of ARGs in selected Asian communities. Set A samples (n = 21) were collected weekly from May to July 2020 at the influent, secondary-treatment, and effluent stages of a WWTP in Japan, whereas Set B samples (n = 40) were collected between 2022 and 2023 from sites in Japan, the Philippines, Vietnam, and Thailand. Samples were analyzed using a high-throughput quantitative polymerase chain reaction on the Biomark HD System, targeting fecal indicators, mobile genetic element, and ARGs. In Set A, all targeted ARGs and int1 were detected, except for vanA. Substantial ARG concentrations persisted in the effluent. Japan and Thailand samples showed ARG patterns more similar to community-associated resistance, whereas Vietnam and the Philippines samples showed patterns potentially influenced by healthcare-associated sources. Improved treatment and targeted surveillance strategies are necessary for managing the environmental dissemination of antimicrobial resistance.