Pierce Cell Surface Biotinylation and Isolation Kit / 细胞膜蛋白生物素化和分离试剂盒
货号：A44390
规格：8 samples
价格：5681
产品类型：蛋白提取/纯化
品牌：Thermo Fisher
| The Thermo Scientific Pierce Cell Surface Protein Biotinylation and Isolation Kit enables the selective biotinylation, solubilization, and enrichment of plasma membrane proteins.This easy-to-use kit provides all of the necessary components and procedure for optimal labeling and subsequent isolation of cell surface proteins. Buffers are supplied pre-formulated to produce consistent results. Mammalian cells are first labeled with EZ-Link Sulfo-NHS-SS-Biotin, a thiol-cleavable amine-reactive biotinylation reagent. Cells are subsequently lysed and the labeled proteins are captured with NeutrAvidin Agarose. Dithiothreitol (DTT) is used in the elution to reduce the disulfide bonds in the biotin label, resulting in the release of the bound proteins without the biotin label. This kit contains sufficient reagents for eight experiments, consisting of two 85-95% confluent 15-cm dishes or four 75-cm2 flasks.Cell surface proteins represent a key subset of cellular proteins and play major roles in signal transduction, cell adhesion, and ion transport. These proteins are challenging to efficiently extract and isolate due to their multiple membrane-spanning domains. Often, the harsh detergents and conditions necessary for efficient extraction result in denaturation and contamination of plasma membrane proteins after isolation. As an alternative, this kit utilizes a membrane-impermeable, amine-reactive, sulfo-NHS-SS-biotin to efficiently label cell surface proteins with accessible lysines. The lysis, capture, wash, and elution conditions have been optimized for efficient enrichment and isolation while minimizing contamination of intracellular proteins.The protocol has been optimized for use with diverse mammalian cell lines, including adherent and suspension cells, and is useful for differential expression analysis between treated and non-treated cells or between one or more cell lines. Selectivity and efficiency of the cell surface protein isolation has been verified by western blotting and LC-MS analysis. |
| 特点： |
| • Improved performance—kit and protocol have been re-designed to improve enrichment and extraction of biotinylated plasma membrane proteins while minimizing intracellular protein contamination• Optimized—protocol has been refreshed to reduce processing time and the number of steps, and reagent formulations optimized for improved performance• Compatible—reagents have been verified with suspension and adherent cell lines, with additional instructions provided for MS sample preparation• Validated applications—reagents and procedures are compatible with western blotting and MS applications |
| 数据： |
| Classification of cell surface protein type in the top 50 most abundant proteinsCell surface proteins were enriched from three mammalian cell lines, in duplicate, and then analyzed by LC-MS. Raw data was searched using Proteome Discoverer Software 2.2. The top 50 most abundant proteins were identified using the UniProt Knowledgebase. Cell surface proteins were classified as single- or multi-spanning (6-17 spans) transmembrane (TM) proteins, glycosylphosphatidylinositol (GPI) anchored proteins, or extracellular matrix (ECM) proteins. A similar distribution was observed in the three cell lines. Protocol summary for the Cell Surface Protein Biotinylation and Isolation Kit Western blot analysis of enriched cell surface proteinsSamples were prepared in duplicate from two cell lines using the Cell Surface Protein Biotinylation and Isolation Kit. Flow-through (F), elution (E), and bead boil (B) were normalized by volume and analyzed by western blot for cell surface proteins (EGFR, CD55, cadherin, and integrin 5) or intracellular proteins (HSP90, actin, calnexin, and -tubulin). The blots were imaged using the Bright FL1000 Imaging System. The data demonstrates effective enrichment of cell surface proteins with minimal contamination of intracellular proteins. Cell surface protein classification in the top 100 most abundant proteinsCell surface proteins from A549 cells were isolated using Cell Surface Protein Biotinylation and Isolation Kit (Cat. No. A44390) or Pierce Cell Surface Protein Isolation Kit (Cat. No. 89881) in duplicate. A549 whole cell lysate was analyzed as a no-enrichment control. Samples were then prepared for MS analysis using the EasyPep Mini MS Sample Prep Kit with modifications indicated in the user manual. Peptide amounts were normalized using the Pierce Quantitative Colorimetric peptide Assay Kit and then analyzed by LC-MS. Raw data was searched using Proteome Discoverer Software version 2.2, and cell surface proteins were identified using the UniProt Knowledgebase. Approximately 5-fold more cell surface proteins and 4-fold fewer intracellular proteins were identified with Cell Surface Protein Biotinylation and Isolation Kit compared with Pierce Cell Surface Protein Isolation Kit. The protein distribution for Pierce Cell Surface Protein Isolation Kit was similar to whole cell lysate. Enhancement of enrichment of high- and low-abundance cell surface proteinsCell surface proteins from A549 cells were isolated using Cell Surface Protein Biotinylation and Isolation Kit (Cat. No. A44390) or Pierce Cell Surface Protein Isolation Kit (Cat. No. 89881) in duplicate. A549 whole cell lysate was analyzed as a no-enrichment control. Samples were then prepared for MS analysis using the EasyPep Mini MS Sample Prep Kit with modifications indicated in the user manual. Peptide amounts were normalized using the Pierce Quantitative Colorimetric peptide Assay Kit and then analyzed by LC-MS. Raw data was searched using Proteome Discoverer Software version 2.2, and cell surface proteins were identified using the UniProt Knowledgebase. Enhanced enrichment of cell surface proteins was observed in extracts prepared using Cell Surface Protein Biotinylation and Isolation Kit when compared to extracts prepared using Pierce Cell Surface Protein Isolation Kit or whole cell lysate. Similar results were observed for proteins ranging in abundance by up to 2 orders of magnitude. |
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