RESUMEN
Herein, you will find detailed information for the preparation of a coarse-grained array of peptides embedded in a lipid membrane. It contains all the steps to set up and run a molecular dynamic simulation using a coarse-grained approach. We provide analytical tools and scripts for generating a residue-level contact matrix between multiple peptides, as well as geometric analysis of arrangements between multiple peptides. This protocol was designed to study the organization of transmembrane peptides in an unbiased manner using computational approaches. For complete details on the use and execution of this protocol, please refer to Smulski et al. (2022).
Asunto(s)
Simulación de Dinámica Molecular , Péptidos , Péptidos/químicaRESUMEN
Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b).
Asunto(s)
Enzimas , Escherichia coli , Proteínas Recombinantes , Cromatografía de Afinidad , Pruebas de Enzimas , Enzimas/genética , Enzimas/aislamiento & purificación , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Tipificación Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transformación BacterianaRESUMEN
Galectins are soluble lectins that participate in many physiological and pathological functions. Since they can act extracellularly, the use of the recombinant protein is a recurrent strategy for studying their biological functions. Here, we provide a general protocol for the production of Galectins and their isolated or chimeric domains. We take advantage of their lectin activity and the 6xHis-tag addition for purification, thus obtaining a highly pure and active Galectin to use in both in vitro and in vivo assays. For complete details on the use and execution of this protocol, please refer to Cattaneo et al. (2011), Tribulatti et al. (2012), and Prato et al. (2020).
Asunto(s)
Cromatografía de Afinidad/métodos , Galectinas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Bacterias/metabolismo , Sitios de Unión , Galectinas/biosíntesis , Hemaglutininas , Lectinas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismoRESUMEN
The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals.