SPDP

Screen protected, drip proof (SPDP) As above, but with additional proteetion against dripping of water Figures 1.18(a)-(d). 

Vo/r SPDP motors also have a cooling fan bul their surface is plain, whereas TEFC motors have fins on their housings that add to their cooling surface. Refer to Figures I.l8(a)-(c) and 1.19(a)and (b). 

Figure 1.18(a) Screen protected drip proof (SPDP) squirrel cage motor (Cooling system ICOA1)... 

SPDP-modified proteins can be reduced with DTT to yield free sulfhydryl groups for conjugation. 

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. 

SMPT, succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene, contains an NHS ester end and a pyridyl disulfide end similar to SPDP, but its hindered disulfide makes conjugates formed with this reagent more stable (Thorpe et al., 1987) (Chapter 5, Section 1.2). The reagent is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 21, Section 2.1). A water-soluble analog of this crosslinker containing an extended spacer arm is also commercially available as sulfo-LC-SMPT (Thermo Fisher). 

SPDP, 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... 

Figure 5.2 SPDP can react with amine-containing molecules through its NHS ester end to form amide bonds. The pyridyl disulfide group then can be coupled to a sulfhydryl-containing molecule to create a cleavable disulfide bond.

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. 

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

Figure 7.9 Amine-containing dendrimers can be activated with SPDP to create thiol-reactive derivatives. Alternatively, the pyridyl dithiol group may be reduced to create free thiols on the dendrimer surface for subsequent conjugation.

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. 

Purify the derivatized dendrimer using gel filtration (size exclusion chromatography) on a desalting column or through use of ultrafiltration spin-tubes (for G-4 and above). For smaller dendrimers, the derivatives may be purified by repeated precipitation from a meth-anolic solution by addition of ethyl acetate, dioxane, or benzene. The SPDP-dendrimer may be dried by lyophilization (if in water or buffer) or by solvent evaporation in vacuo (if the precipitation method was used). 

Dissolve the purified SPDP-modified dendrimer of step 5 in 50 mM sodium phosphate, 0.15M NaCl, pH 7.5, or in DMSO at a concentration of at least lOmg/ml. Add a 10-20 X molar excess of an amine-reactive fluorescent molecule (i.e., NHS-rhodamine or a hydrophilic NHS-Cy5 derivative see section on fluorescent probes). React with mixing for 1 hour at room temperature. Purify the fluorescently labeled SPDP-modified dendrimer using gel filtration or ultrafiltration. Follow the method of either step 7 or 8 to conjugate the dendrimer to another protein or molecule. 

Alternatively, a thiol-containing protein may be directly conjugated to the SPDP-modified dendrimer to create a disulfide linkage. Add a sulfhydryl-containing protein... 

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