General description

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 â€“ 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 Benefits

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

Caution

This product is sensitive to moisture. The vial is packaged in a resealable bag with a desiccant to reduce exposure to moisture. After cold storage, equilibrate the vial to room temperature before opening to reduce condensation inside the vial. Make fresh solutions. Storage of stock solutions is not recommended. After use, return the vial to the resealable bag. Close the bag and store the product at the recommended temperature.