Heterobifunctional Crosslinkers

N-succinimidyl-3-(2-pyridyldithio)propionate, is one of the most popular heterobifunctional crosslinking agents available. The activated NHS ester end of SPDP reacts with amine 

Dissolve a protein or macromolecule containing primary amines at a concentration of 10 mg/ml in 50 mM sodium phosphate, 0.15 M NaCl, pH 7.2. Other non-amine-containing buffers such as borate, HEPES, and bicarbonate also may be used in this reaction. Avoid sulfhydryl-containing components in the reaction mixture as these will react with the pyridyl disulfide end of SPDP. The effective pH for the NHS ester modification reaction is in the range of 7-9, but hydrolysis will increase at the higher end of this range. 

Add 25 pi of the stock solution of either SPDP or LC-SPDP in DMSO to each ml of the protein to be modified. If sulfo-LC-SPDP is used, add 50 pi of the stock solution in water to each ml of protein solution. 

Mix and react for at least 30 minutes at room temperature. Longer reaction times, even overnight, will not adversely affect the modification. 

Purify the modified protein from reaction by-products by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. Alternatively, centrifugal spin columns containing a desalting resin may be used for rapid purification (Thermo Fisher). 

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Haptens with an amino group. Amine groups in haptens, carrier proteins or both can be modified for conjugation through homo- or heterobifunctional crosslinkers such as acid anhydrides (e.g., succinic anhydride), diacid chlorides (e.g.. 

The greatest degree of control in crosslinking procedures is afforded using heterobifunctional reagents (Chapter 5). Since a heterobifunctional crosslinker has different reactive groups on either end of the molecule, each side can be directed specifically toward different functional... 

Figure 1.25 Heterobifunctional crosslinking agents used in multi-step protocols result in the best control over the products formed.

Purify the reduced IgG from excess 2-mercaptoethylamine and reaction by-products by dialysis or gel filtration using a desalting resin. All buffers should contain 1-10 mM EDTA to preserve the free sulfhydryls from metal-catalyzed oxidation. The sulfhydryl-containing half antibody now may be used in conjugation protocols that use —SH-reactive heterobifunctional crosslinkers (Chapter 5, Section 1). 

BMPA is N-(3-maleimidopropionic acid (or 3-maleimidopropionic acid), which contains a thiol-reactive maleimide group at one end and a carboxylate group on the other end (Rich et al., 1975 Moroder, 1983, 1987). The compound is the acid precursor to the short, heterobifunctional crosslinker 3-maleimidopropionic acid N-hydroxysuccinimide ester (BMPS). 

Figure 5.1 The general design of a heterobifunctional crosslinking agent includes two different reactive groups at either end and an organic cross-bridge of various length and composition. The cross-bridge may be constructed of chemically cleavable components for selective disruption of conjugates.

SANPAH (N-succinimidyl-6-(4 -azido-2 -nitrophenylamino)hexanoate) is a heterobifunctional crosslinking agent containing an NHS ester and a photoreactive phenyl azide group (Thermo Fisher). The NHS ester end can react with amine groups in proteins and other molecules, forming... 

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