Bednarska Izabela, Malycheva Darina, Kristensson Maria Alvarado
Molecular Pathology, Department of Translational Medicine, Lund University, Inga Marie Nilssons gata 53, SE-214 28 Malmö, Sweden.
MethodsX. 2024 Jul 11;13:102860. doi: 10.1016/j.mex.2024.102860. eCollection 2024 Dec.
In conventional cell lysate protocols, cell debris is typically discarded to obtain a cleaner lysate. However, this approach has limitations, as it may overlook vital cellular components. By discarding cell debris, researchers may inadvertently exclude crucial elements. Retaining all cellular components offers several advantages for studying molecular biology within various cellular compartments. Firstly, it provides a more accurate representation of the cellular environment. Secondly, it enables the study of complex cellular interactions, including those involving cellular structures and signaling pathways associated with debris. This shift in perspective highlights the importance of a holistic approach to lysate preparation. By obtaining lysates that include all cellular components, researchers can gain deeper insights into cellular processes, leading to more accurate data and a better understanding of cellular function and dysfunction. This study aimed to develop a protocol for the preparation of total cell lysates that retain all cellular components, including debris. Our method involves:•A three-step solubilization process using a combination of detergents, saccharides, and chelators, coupled with sonication, in contrast to the classical one-step approach using an all-detergent cocktail.•A comprehensive strategy ensuring the solubilization of all cellular components, providing a more complete lysate for analysis.
在传统的细胞裂解物制备方案中，通常会丢弃细胞碎片以获得更纯净的裂解物。然而，这种方法存在局限性，因为它可能会忽略重要的细胞成分。通过丢弃细胞碎片，研究人员可能会无意中排除关键要素。保留所有细胞成分对于研究不同细胞区室中的分子生物学具有诸多优势。首先，它能更准确地呈现细胞环境。其次，它有助于研究复杂的细胞相互作用，包括那些涉及与碎片相关的细胞结构和信号通路的相互作用。这种观念的转变凸显了采用整体方法进行裂解物制备的重要性。通过获得包含所有细胞成分的裂解物，研究人员能够更深入地了解细胞过程，从而获得更准确的数据，并更好地理解细胞功能和功能障碍。本研究旨在开发一种制备保留所有细胞成分（包括碎片）的全细胞裂解物的方案。我们的方法包括：• 与使用全洗涤剂混合物的经典一步法不同，采用三步溶解过程，使用洗涤剂、糖类和螯合剂的组合，并结合超声处理。• 一种全面的策略，确保所有细胞成分的溶解，为分析提供更完整的裂解物。
