To prepare 4X Laemmli Sample Buffer with reducing agent, add 100 µL of 2-mercaptoethanol to 900 µL of 4X Laemmli Sample Buffer, or add dithiothreitol to 4X Laemmli Sample Buffer to a final concentration of 50 mM. When uncertain, test 4X Laemmli Sample Buffer with and without reducing reagent. EV markers other than CD9/CD63/CD81/CD45/EpCAM may require a reducing agent added to Laemmli Sample Buffer. Note: When blotting for CD9, CD63, CD81, EpCAM, and CD45, do not add a reducing agent to the Laemmli Sample Buffer, as CD9/CD63/CD81 detection antibodies often recognize the disulfide bond on the antigen's epitope. Here, we provide a detailed protocol for performing a western blot to detect the presence of such characteristic EV-associated proteins in order to confirm the presence of EVs in the biological sample. Tetraspanin markers such as CD9, CD63, and CD81 are proteins commonly found on EVs across different cell types. Isolated EVs can be characterized by western blotting (also referred to as immunoblotting), a widely used technique to detect specific protein markers in a sample. Once isolated, the particles should be analyzed to confirm that they are indeed EVs and not products of cell fragmentation or other contaminants such as protein complexes or lipoproteins, usually present in biological samples. Using EasySep™ Human Extracellular Vesicle Positive Selection Kits or Extracellular Vesicle SEC Columns, researchers can easily isolate and purify human EVs from biofluids-including serum and plasma-and from culture-conditioned media. MesenCult™-ACF Plus Medium), such as immunomagnetic separation, differential ultracentrifugation, or size exclusion chromatography (SEC)*. There are several ways for isolating EVs from biofluids or cell culture conditioned media (e.g. Due to their inherently heterogeneous nature as well as the complexity of biological samples, it is recommended to characterize EVs after isolation. These vesicles, carrying protein and genetic cargo, play an important role in intercellular communication and are recognized for their potential therapeutic applications. Tissue and Cell Culture Dissociation ReagentsĮxtracellular vesicles (EVs) are lipid bilayer-enclosed structures released by almost all cell types.Learn from the experts.Ī free, sterile, and safe tool that allows for removal of bands from gels.Work at STEMCELL View Current Opportunities > Introducing the Western Blot Learning Center.The 2-D Doctor is a self-help guide developed by Bio-Rad that enables you to identify and troubleshoot your 2-D gel issues. Troubleshooting 2-D Electrophoresis Gels with 2-D Doctor.Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look their best. Western Blot Doctor Troubleshooting Guide.This guide describes the experimental methods and tools used in 2-D electrophoresis and proteomics research.ĭetails on blotting technology, available products, and methods, plus tips, techniques, and troubleshooting.Ī guide to polyacrylamide gel electrophoresis and protein detection, including theory, product selection, protocols, and more. 2-D Electrophoresis Workflow How-To Guide.Loading buffers for protein or nucleic acid samples, detergents, ampholytes, and gel-casting buffers.įilter by your laboratory set-up and reagents to get a custom western blotting protocol that best fits your needs. Visualize and quantify protein bands including Coomassie stains, fluorescent stains, silver stains, reversible negative stains, and total protein blot stains. Molecular weight standards for applications including SDS-PAGE, western blotting, 2-D PAGE, and isoelectric focusing (IEF). Protein Ladders and Standards (Markers).In-Cell Western Assays are high-throughput quantitative and qualitative immunofluorescence assays that utilizes Bio-Rad's ChemiDoc MP Imaging System and Image Lab Software.ĭetect, image and quantify multiplex fluorescence, chemiluminescence, stain-free and colorimetric blots and gels. Western blotting products include protein transfer systems, membranes, filter paper, blotting buffers, and detection kitsīio-Rad's fluorescent western blotting workflow is a seamless integration of products designed to work together to offer guaranteed results. Our electrophoresis chambers enable rapid, high-resolution protein separation on precast or handcast gels over a variety of different gel sizes.Ĭhoose SDS-PAGE and native-PAGE gels, convert to TGX Precast Gels, or choose specialized gel chemistries including stain-freeįrom basic to high-voltage capacity, our power supplies provide simple programming in a compact, stackable case. Protein Electrophoresis Equipment and Consumables
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