SPDP 试剂的胺反应部分是 N-羟基琥珀酰亚胺（NHS）酯。反应最通常在 pH 7-8 的磷酸盐、碳酸盐/碳酸氢盐或硼酸盐缓冲液中进行。可以使用其他不包含伯胺（或硫醇或二硫化物还原剂–参见巯基反应性的讨论）的缓冲剂。pH 值越高，反应和水解降解的速率越大；例如，NHS 酯的半衰期在 pH 值为 7 时为数小时，而在 pH 值为 9 时不到 10 分钟。SPDP 和 LC-SPDP 等 NHS 酯试剂的水溶性有限，在将其添加到反应混合物之前，必须先将其溶于有机溶剂中。诸如 Sulfo-LC-SPDP 的 Sulfo-NHS-酯试剂是水溶性的，可以直接添加到水性反应混合物中。 特点和优势： 反应性基团：NHS 酯和吡啶基二硫醇 对以下分子具有反应性：氨基和巯基 与游离巯基反应时释放出可检测的副产物；通过测量 343 nm 处吡啶-2-硫酮的释放，可以轻松地跟踪反应进度 间隔臂中的二硫键在pH 8.5条件下易于被10-50mM DTT或TCEP裂解 减少 SDS-PAGE 上样缓冲液，也易于使间隔臂裂解 使用可裂解交联剂，能够实现交联产物的分离 水不溶性（先溶于 DMF 或 DMSO） SPDP 交联剂可透过膜，因此可以在细胞内部进行交联 与其他品种的 SPDP 型试剂进行比较，包括聚乙二醇化形式 The amine-reactive portion of SPDP reagents is the N-hydroxysuccinimide (NHS) ester. Reactions are most commonly performed in phosphate, carbonate/bicarbonate, or borate buffers at pH 7-8. Other buffers can be used provided they do not contain primary amines (or thiols or disulfide reducing reagents – see discussion of the sulfhydryl reactivity). The rate of reaction and degradation by hydrolysis increases with increasing pH; for example, the half-life of the NHS ester is several hours at pH 7 and less than 10 minutes at pH 9. NHS-ester reagents like SPDP and LC-SPDP have limited aqueous solubility and must be dissolved in organic solvent before adding them to a reaction mixture. Sulfo-NHS-ester reagents like Sulfo-LC-SPDP are water-soluble and can be added directly to aqueous reaction mixtures. Features and advantages: Reactive groups: NHS ester and pyridyldithiol Reactive towards: amino and sulfhydryl groups Releases a detectable by-product when reacted with a free sulfhydryl group; by measuring the release of pyridine-2-thione at 343 nm, the reaction can be easily followed Disulfide bond in the spacer arm is readily cleaved by 10-50 mM DTT or TCEP at pH 8.5 Spacer arm is also easily cleaved using reducing SDS-PAGE sample loading buffer Cleavable crosslinker allows separation of crosslinked products Water-insoluble (dissolve first in DMF or DMSO) SPDP crosslinker is membrane permeable, so crosslinking can be done inside the cells Compare to other varieties of SPDP-type reagents, including pegylated forms