General Method for PEG PFP Ester Conjugation

聚乙二醇酯共轭的通用方法

I. Introduction

The PEG PFP Ester is a class of PEG labeling reagents that react with primary ａnd secondary amines. The pentafluorophenyl (PFP) ester is less subject to hydrolysis than NHS esters, resulting in more efficient reactions. PEG PFP Ester must first be dissolved in a minimal amount of an organic solvent, such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF) ａnd then added to the buffer containing the protein or other molecule. The reagent forms an emulsion that allows the reaction to proceed.

一、介绍

PEG-PFP酯是一类与伯胺和仲胺反应的PEG标记试剂。五氟苯基（PFP）酯比NHS酯更容易水解，从而产生更有效的反应。PEG PFP酯必须先溶解在少量有机溶剂中，如二甲基亚砜（DMSO）或二甲基甲酰胺（DMF），然后添加到含有蛋白质或其他分子的缓冲液中。该试剂形成一种乳液，使反应继续进行。

II. Product Information

• PEG PFP Ester is moisture-sensitive. Store the vial of reagent at -20°C with desiccant. To avoid moisture condensation onto the product, equilibrate vial to room temperature before opening.

• As directed in the procedure, dissolve the PEG PFP Ester reagent immediately before use. The PFP moiety readily hydrolyzes ａnd becomes non-reactive; therefore, weigh ａnd dissolve only a small amount of the reagent at a time, ａnd do not prepare stock solutions for storage. Discard any unused reconstituted reagent.

• Avoid buffers containing primary amines (e.g., Tris or glycine) as these will compete with the reaction. If necessary, dialyze or desalt to exchange the protein sample into an amine-free buffer such as phosphate-buffered saline (PBS).

• During the PEGylation process, unreacted linker is easily removed by size exclusion using either desalting columns or dialysis. A 10mL desalting column is best suited for processing PEGylation reactions involving 1-10mg of protein in approximately 0.5-2mL. For smaller amounts of protein and/or smaller reaction volumes, both the PEGylation reaction ａnd subsequent buffer exchange may be performed in a single Thermo Scientific Slide-A-Lyzer MINI Dialysis Unit.

二 产品信息
PEG PFP酯对水分敏感。用干燥剂将试剂瓶存放在-20°C。为避免水分凝结到产品上，打开将小瓶平衡到室温。
按照程序中的指示，在使用立即溶解PEG PFP酯试剂。PFP部分容易水解并变得不反应；因此，一次只称量和溶解少量试剂，不准备储备溶液进行储存。丢弃任何未使用的重组试剂。
避免使用含有伯胺（如三胺或甘氨酸）的缓冲液，因为它们会与反应竞争。如有必要，透析或脱盐，将蛋白质样品交换成无胺缓冲液，如磷酸盐缓冲盐（PBS）。
在聚乙二醇化过程中，未反应的连接剂很容易通过使用脱盐柱或透析排除而去除。10毫升脱盐柱适合于处理含1-10毫克蛋白质、约0.5-2毫升的聚乙二醇化反应。对于少量蛋白质和/或较小的反应体积，聚乙二醇化反应和随后的缓冲液交换可在单一的热学中进行。Slide-A-Lyzer微型透析装置。

III. Additional Materials Required

• Phosphate-buffered Saline (PBS): 0.1M phosphate, 0.15M sodium chloride; pH 7.2 or other non-amine containing buffer at pH 7.0-8.0

• Quenching Buffer: Tris-buffered saline (TBS; 25mM Tris, 0.15M sodium chloride; pH 7.2; glycine or other amine-containing buffer)

• Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF)

• 10-100 μL sample volumes; Slide-A-Lyzer® Dialysis Cassette Kit for 0.1-30.0mL sample volumes; or Zeba Spin Desalting Columns for sample volumes ranging from >10μL to 4mL

三、所需附加材料
磷酸盐缓冲盐（PBS）：0.1 m磷酸盐，0.15 m氯化钠；pH7.2或其他非含胺缓冲液，pH7.0-8.0
缓冲液：Tris缓冲盐水（TBS；25 mm Tris，0.15 m氯化钠；pH7.2；甘氨酸或其他含胺缓冲液）
水溶性有机溶剂，如二甲基亚砜（DMSO）或二甲基甲酰胺（DMF）
10-100μl样品体积；0.1-30.0ml样品体积的Slide-A-Lyzer透析盒套件；或样品体积大于10μl至4ml的Zeba旋转脱盐柱。

聚乙二醇酯共轭的通用方法

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