BioTechniques最新文献_第2页

A simple and rapid agarose gel electrophoresis method to assess CpG methylation of DNA. 一种简单快速的琼脂糖凝胶电泳测定DNA CpG甲基化的方法。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-04-20 DOI: 10.1080/07366205.2026.2656194

Marco Paliza-Carre, Ge Chen, Shahrokh Shabahang

{"title":"A simple and rapid agarose gel electrophoresis method to assess CpG methylation of DNA.","authors":"Marco Paliza-Carre, Ge Chen, Shahrokh Shabahang","doi":"10.1080/07366205.2026.2656194","DOIUrl":"10.1080/07366205.2026.2656194","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"170-182"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147728224","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

3in1: an ultra-fast phage display biopanning method overcoming the biopanning bias and yielding more diverse binders. 3in1：一种超快速噬菌体显示生物筛选方法，克服了生物筛选的偏见，产生了更多样化的结合物。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-02-28 DOI: 10.1080/07366205.2026.2629753

Samson Lichtenstein, Morgane Valles, Michael Mullin, Aleardo Morelli

引用次数: 0

A robust and sensitive method for detecting subtle structural differences in bovine serum albumin. 一种检测牛血清白蛋白细微结构差异的稳健灵敏方法。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-03-03 DOI: 10.1080/07366205.2026.2635455

Teruo Akuta, Tomomi Sato, Masataka Nakagawa, Yuki Komatsu, Tsutomu Arakawa, Taiji Oyama

{"title":"A robust and sensitive method for detecting subtle structural differences in bovine serum albumin.","authors":"Teruo Akuta, Tomomi Sato, Masataka Nakagawa, Yuki Komatsu, Tsutomu Arakawa, Taiji Oyama","doi":"10.1080/07366205.2026.2635455","DOIUrl":"10.1080/07366205.2026.2635455","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"65-80"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147343282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Investigating the interactomic landscape of survival motor neuron (SMN) and the SMNΔ7 truncated protein. 研究存活运动神经元（SMN）和SMNΔ7截短蛋白的相互作用景观。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-06-17 DOI: 10.1080/07366205.2026.2675476

Bobby Beaumont, Silvia A Synowsky, Sally L Shirran, Judith E Sleeman

{"title":"Investigating the interactomic landscape of survival motor neuron (SMN) and the SMNΔ7 truncated protein.","authors":"Bobby Beaumont, Silvia A Synowsky, Sally L Shirran, Judith E Sleeman","doi":"10.1080/07366205.2026.2675476","DOIUrl":"https://doi.org/10.1080/07366205.2026.2675476","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"207-220"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148263171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Why and when should we choose digital PCR? 我们为什么以及何时应该选择数字PCR？

IF 2.5 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-06-03 DOI: 10.1080/07366205.2026.2678320

Mustafa Ahmad Munawar, Sofie Mohlin

引用次数: 0

Self-circularized Overhang-based Unrecombined Products (SOUP): a rapid in vitro method for generating backbone-free expression cassettes. 基于自循环悬垂的非重组产物（SOUP）：一种快速生成无主干表达盒的体外方法。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-03-14 DOI: 10.1080/07366205.2026.2645351

Ionuț Adrian Cepleanu-Pascu, Mirel Adrian Popa, Gheorghe Dănuț Cimponeriu, Ileana Stoica

{"title":"Self-circularized Overhang-based Unrecombined Products (SOUP): a rapid in vitro method for generating backbone-free expression cassettes.","authors":"Ionuț Adrian Cepleanu-Pascu, Mirel Adrian Popa, Gheorghe Dănuț Cimponeriu, Ileana Stoica","doi":"10.1080/07366205.2026.2645351","DOIUrl":"10.1080/07366205.2026.2645351","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"81-92"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147455666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation and validation of C-terminal LRP8 antibodies for detecting processed intracellular fragments. 用于检测加工过的细胞内片段的c端LRP8抗体的产生和验证。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-03-31 DOI: 10.1080/07366205.2026.2645347

Alessandro Medoro, Emanuele Foderà, Maurizio Ronci, Marco Trerotola, Donatella Mignogna, Gennaro Raimo, Mariano Intrieri, Anna De Martino, Alessio Lepore, Carola Porcile, Teresa Benincasa, Claudio Russo, Daniela Passarella

{"title":"Generation and validation of C-terminal LRP8 antibodies for detecting processed intracellular fragments.","authors":"Alessandro Medoro, Emanuele Foderà, Maurizio Ronci, Marco Trerotola, Donatella Mignogna, Gennaro Raimo, Mariano Intrieri, Anna De Martino, Alessio Lepore, Carola Porcile, Teresa Benincasa, Claudio Russo, Daniela Passarella","doi":"10.1080/07366205.2026.2645347","DOIUrl":"10.1080/07366205.2026.2645347","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"111-122"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147589824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A refined qPCR duplexing method for the molecular sex determination of sturgeon (Acipenser genus), providing a validating, endogenous control. 一种用于鲟鱼（Acipenser属）分子性别测定的改进的qPCR双链法，提供了一种有效的内源性对照。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-09-03 DOI: 10.1080/07366205.2026.2723752

Britton Ranson Olson, Alana J Miller-Schofield, Kevin L Kapuscinski

{"title":"A refined qPCR duplexing method for the molecular sex determination of sturgeon (Acipenser genus), providing a validating, endogenous control.","authors":"Britton Ranson Olson, Alana J Miller-Schofield, Kevin L Kapuscinski","doi":"10.1080/07366205.2026.2723752","DOIUrl":"https://doi.org/10.1080/07366205.2026.2723752","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"303-310"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148886385","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Single-cell RNA-sequencing of air-liquid interface cultures: a practical approach. 气液界面培养的单细胞rna测序：一种实用的方法。

IF 2.5 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-03-28 DOI: 10.1080/07366205.2026.2621052

Firoozeh Vosoogh-Gerayeli, Carolyn J Wang, Chen X Yang, Gurpreet S Singhera, Edward Z Li, Xuan Li, Julia S W Yang, Chung Y Cheung, Janice M Leung, Don D Sin

引用次数: 0

Label-free DNA-sensor modelling based on magnetic induction spectroscopy. 基于磁感应光谱的无标记dna传感器建模。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-03-27 DOI: 10.1080/07366205.2026.2647885

César A González-Díaz, Boris Rubinsky, Simo A Mäkiharju, Virginia Sánchez-Monroy, Franck M André, Luis M Mir

引用次数: 0