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
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