SIAB

Conjugations with iodoacetyl crosslinkers have been done using ricin and CVF (Thorpe et al., 1984 Vogel, 1987 Myers et al., 1989). The following generalized protocol for using SIAB is based on the method of Cumber et al. (1985). 

Caution toxin molecules are dangerously toxic even in small amounts. Use extreme care in handling. 

To prepare an antibody-ricin conjugate using this protocol, 2.25 mg of antibody is needed 

Dissolve intact ricin in 0.1M sodium phosphate, 0.15M NaCl, pH 7.5, at a concentration of lOmg/ml. 

Dissolve SIAB (Thermo Fisher) in DMSO at a concentration of 1.4mg/ml. Prepare fresh and protect from light to avoid breakdown of the active halogen group. 

Add 160 xl (225 p,g) of the SLAB solution to each milliliter of the ricin solution. 

---

Thus for equal-size spheres the force between them is just xn f/siab-siab(J ) d is directly related to the potential energy between two slabs [13]. This point is examined further in the problems at the end of the chapter. 

Fig. 4.8 Enhancement of interaction potential in a siit-shaped pore between paraliel siabs of solid. Plot of 0/0 against z/rg for various values of d/r or R/r (see text). (Reduced from a diagram of Everett and Fowl. )...

SIAB Sachsisches Institut fiir Angewandte Biotechnologie e.V. an der Universitat Leipzig, Permoserstrafie 15,04318 D-Leipzig, Germany... 

Since SIAB is water-insoluble, it must be dissolved first in organic solvent prior to addition to an aqueous reaction medium. The most commonly used solvents for this purpose include DMSO and DMF. Typically, a concentrated stock solution is prepared in one of these solvents and an aliquot added to the protein conjugation solution. Long-term storage of the reagent in these solvents is not recommended, however, due to slow uptake of water and breakdown of the NHS ester end. 

Conjugations done with SIAB usually proceed by a multi-step process. Because the crosslinker s NHS ester end is its most labile functionality, an amine-containing protein or molecule... 

Figure 5.6 SIAB may be used to modify an amine-containing molecule for subsequent conjugation to a sulfhydryl-containing molecule.

SIAB and sulfo-SIAB have been used to make a high-capacity RNA affinity column for the purification of human IRP1 and IRP2 (Allerson et al., 2003), to couple antibodies or Fab fragments to amine-modified microparticles (Harma et al., 2000), and in the attachment of oligonucleotides to surfaces for detection arrays (Adessi et al., 2000). 

The following protocol illustrates the use of SIAB in preparing antibody-enzyme conjugates using P-galactosidase. 

Add 100 pi of the SIAB stock solution to each ml of the antibody solution. Mix gently to dissolve. 

Conjugation reactions done with SIAC or SIACX are usually multi-step procedures similar to the protocol described for SIAB, previously. 

Figure 21.12 SIAB can be used to activate toxin molecules for coupling with sulfhydryl-containing antibodies. In this case, the antibody molecule is thiolated using SATA and deprotected to reveal the free sulfhydryl. Reaction with the SIAB-activated toxin forms the final conjugate by thioether bond formation.

Concentrate the purified, SIAB-activated toxin to lOmg/ml using centrifugal concentrators with a MW cutoff of 10,000. Protect the activated toxin from light to prevent degradation of the iodoacetyl-reactive group. 

Conjugation of SIAB-Activated Toxin with Thiolated Antibody... 

The most common type of heterobifunctional reagent used for the activation of lipid components includes the amine- and sulfhydryl-reactive crosslinkers containing an N-hydroxysuccinim-ide (NHS) ester group on one end and either a maleimide, iodoacetyl, or pyridyl disulfide group on the other end (Chapter 5, Section 1). Principle reagents used to effect this activation process include SMCC (Chapter 5, Section 1.3), MBS (Chapter 5, Section 1.4), SMPB (Chapter 5, Section 1.6), SIAB (Chapter 5, Section 1.5), and SPDP (Chapter 5, Section 1.1). Other... 

Figure 22.29 SIAB-activated liposomes can couple with sulfhydryl-containing proteins to produce thioether linkages.

---