蛋白质浓缩和缓冲液置换
蛋白质浓缩和缓冲液置换常用于蛋白质和抗体纯化、生物工艺和蛋白质组学分析流程。蛋白质、酶、抗体和病毒等大分子溶液的样品制备过程通常会使缓冲液产生大量与下游工艺或检测不兼容的稀释蛋白质和其他生物分子。提取样品后，通常需在分析或使用浓缩和澄清稀释蛋白质。
蛋白质浓缩
现有多种蛋白质浓缩方法，包括多孔滤膜过滤、高渗溶液透析和沉淀/盐析。超滤法相对便捷，在压力或离心力作用下借助半透膜浓缩蛋白，蛋白质变性、失活和降解程度极低。利用此技术，可让过量液体流经滤膜，截留下较大的蛋白质分子，通过缩减样品体积提高蛋白浓度。
蛋白质富集
所谓蛋白质富集，指浓缩生物样品中特定种类或亚型的蛋白质以便进一步分析。蛋白质富集法用于分离低丰度蛋白，可降低样品复杂程度。为改善下游蛋白质组学分析，富集过程会分离并浓缩靶标蛋白质。蛋白质富集过程常基于蛋白质分子量差异，利用超滤法分离蛋白质。
缓冲液置换和脱盐
纯化的蛋白质往往需要转移到合适的缓冲液中才可进一步分析。缓冲液置换、脱盐和去垢剂去除可采用以下方法：
- 透析：基于透膜能力去除渗透性小分子，如盐、去垢剂、溶剂和其他杂质。
- 柱层析：利用体积排阻法凝胶过滤柱去除盐分。
- 渗滤：在压力或离心力作用下，基于分子大小，借助多孔超滤膜去除盐、去垢剂和其他杂质。
相关技术文章
- Large-volume centrifugal filter devices are a convenient alternative to stirred cell ultrafiltration devices for purifying proteins from large volumes of solution.
- Large-volume centrifugal filter devices are a convenient alternative to stirred cell ultrafiltration devices for purifying proteins from large volumes of solution.
- Desalting at laboratory scale is a well-proven, simple, and fast method that will rapidly remove low molecular weight contaminants at the same time as transferring the sample into the desired buffer in a single step.
- The Amicon® Stirred Cell family of pressure-driven filtration devices provide an ideal solution for concentrating and buffer-exchanging large volumes of macrosolutes. These devices are available in multiple sizes to offer a wide range of processing volumes.
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相关实验方案
- These tables will help you navigate the preparation of many common buffer solutions by pH and pKa. Choose a buffer with an effective pH range for your specific assay.
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- User guide for Amicon® Stirred cells including features, operation, specifications, and troubleshooting.
- Detergents are frequently used to solubilize proteins and nucleic acids during purification, but the presence of detergents may interfere with downstream analyses.
- If an antibody must be purified from a small sample volume (0.5-10 mL), then the Amicon® Pro Purification System is recommended.
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