Preparation of Spleen Cells

Instruments required are medium-sized scissors, fine scissors and fine forceps. They may be adequately sterilized by boiling in water for 5 min or by autoclaving. The animal may be killed by cervical dislocation or asphyxiation in C02. (The former is instantaneous, and therefore preferable). The animal should then be swabbed with 70 ethanol, the superficial skin pinched up over the left side of the abdomen, and a small cut made over the spleen. The skin is then torn back, revealing the...

Procedure for Coupling of Kphycoerythrin to Antibodies

This procedure is slightly modified from that of R. Haugland, as described in a brochure from Molecular Probes (see also Haugland, 1995). It proceeds in three basic steps. First, the antibody is derivatized with a succinimidyl ester maleimide derivative such as SMCC (Molecular Probes, cat. no. S-1534). This reacts with lysine residues by the same mechanism as biotin succinimide ester (see Section 10.3.1), and adds thiol-reactive maleimide groups to the antibody. The second step produces...

Binding of Rabbit Sheep and Goat IgG to Staphylococcal Protein A and Streptococcal Protein G

The binding of IgG to protein A has been discussed in Sections 5.3.5 and 9.3.2. Rabbit antisera have the distinct advantage that all their IgG binds to protein A at pH 7.4, and all elutes at pH 4.0. This fact may be used to purify rabbit IgG, and to separate Fab and F(ab')2 fragments of rabbit IgG from Fc or undigested IgG (Goding, 1976, 1978). Affinity chromatography on protein A is the method of choice for rabbit antisera. Sheep and goats have two IgG subclasses. The major subclass (IgGl)...

Proteins that are Very Similar to Self Immune Response Genes

Some proteins are much more highly conserved than others. For example, his-tones are among the most highly conserved proteins, and their amino acid sequence varies little between species. As the amino acid sequence of proteins becomes closer and closer to self, they tend to become less and less immunogenic, and gaps in the repertoire become more and more apparent. The limiting of the repertoire owing to tolerance in the case of antigens that are very similar to self reveals another level of...

Radiolabelling of Monoclonal Antibodies

Radiolabelling of antibodies involves the same principles and procedures that apply to proteins in general, and the reader is referred to Chapter 10 for a more detailed discussion. Antibodies may be radioiodinated to high specific activity with l25I by the chloramine-T method (Section 10.4.1). Incorporation of one iodine per IgG molecule corresponds to a specific activity of approximately 12 pCi jig. Experience with affinity-purified polyclonal antibodies from goat, sheep or rabbit indicates...

Solubilization of Membrane Proteins

Membrane Protein Solubilization

Before the classical techniques of biochemical purification and analysis can be applied to membrane proteins, they must be converted into a water-soluble form. In a few limited and special cases, solubilization of membrane proteins may be achieved by detachment from the membrane using proteolytic cleavage, or relatively minor changes in ionic conditions. In almost all other cases, however, the solubilization of membrane proteins in intact and native form can only be achieved by the use of...

Western Blots

Immunoprecipitation analysis, as described in earlier portions of this chapter, has some fundamental limitations. It is seldom possible to know with certainty whether a radioactive band on a gel represents the polypeptide recognized by the antibody, or whether it is precipitated because it is physically attached to another (possibly nonradioactive) molecule which bears the antigenic determinant, A second problem with immunoprecipitation is that it may be difficult to radiolabel the antigen to...

Fragmentation of Monoclonal Antibodies

Antibody Fragmentation Ficin

In certain circumstances, it may be desirable to generate antigen-binding fragments of monoclonal antibodies. For example, when cells with receptors for the Fc portion of IgG are present (e.g. macrophages, monocytes), intact IgG molecules may bind nonspecifically via their Fc portions (see Section 5.3.4). Removal of the Fc will prevent this mode of binding. The production of small fragments of IgM (intact Mr 900000) may aid penetration into the tissues for cytochemical studies. The older...