LC-SPDP

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. 

Modification of Amine-Dendrimers with Suifo-NHS-LC-SPDP... 

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

Dissolve sulfo-NHS-LC-SPDP at a concentration of 20mM (5.2mg/ml) in DMSO or water. If water is used, the solution must be used immediately to prevent hydrolysis of the sulfo-NHS ester. 

LC-SPDP (Chapter 5, Section 1.1) is an analog of SPDP containing a hexanoate spacer arm within its internal cross-bridge. The increased length of the extended crosslinker is important in some conjugations to avoid steric problems associated with closely linked macromolecules. However, for the preparation of immunotoxins, no advantages were observed for LC-SPDP over SPDP (Singh et al., 1993). 

N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) also may be used to activate pure PE lipids in a similar manner to SMPB. The result will be a derivative containing pyridyl disulfide groups rather than maleimide groups (Figure 22.12). Pyridyl disulfides react with sulfhydryls to form disulfide linkages. Either the standard SPDP or the long-chain version, LC-SPDP, may be employed in the following protocol. 

Add 50 pi of the SPDP (or LC-SPDP) solution to the oligo solution. Add 100 pi of the sulfo-LC-SPDP solution, if the water-soluble crosslinker is used. Mix. 

Dissolve SPDP at a concentration of 6.2 mg/ml in DMSO (makes a 20 miVl stock solution). Alternatively, LC-SPDP may be used and dissolved at a concentration of 8.5 mg/ml in DMSO (also makes a 20 mM solution). If the water-soluble Sulfo-LC-SPDP is used, a stock solution in water may be prepared just prior to addition of an aliquot to the thiolation reaction. In this case, prepare a 10 mM solution of Sulfo-LC-SPDP by dissolving 5.2 mg/ml in water. Since an aqueous solution of the cross-linker will degrade by hydrolysis of the sulfo-NHS ester, it should be used quickly to prevent significant loss of activity. If a sufficiently large amount of protein will be modified, the solid may be added directly to the reaction mixture without preparing a stock solution in water to allow accurate weighing of Sulfo-LC-SPDP. 

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