By Graham J.M., Higgins J.A.
This moment quantity of protocols bargains the main finished set of contemporary analytical concepts on hand for learning the structure and serve as of membranes. It gains the appliance of biochemical, spectroscopic, and fluorimetric ways to the research of molecular topology, the dynamic facets of membrane constitution, and ion delivery. Antibody know-how, research of molecules interested in intracellular signaling, and receptor-ligand interactions also are coated.
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Extra info for Biomembrane Protocols. Architecture and Function
Use of sulfo-MBS avoids exposure of the carrier protein to organic solvents, such as dimethylformamide, that are needed to dissolve the widely used water-insoluble crosslmker m-maleimidobenzoyl-Nhydroxysuccinimide ester (MBS). Such exposure frequently leads to precipitation of the carrier protein, especially if the latter is keyhole limpet hemocyanin. Although the use of sulfo-MBS avoids this problem, tt does not avoid a second potential problem, which is that the S-maleimidobenzoyl linkage is m itself immunogemc.
Pool the peak fractions, rapidly neutralize with OSM acetic acid, then dialyze against PBS. Store the dialyzed IgG at -70°C. 02% (w/v) sodium azide at 4°C. 4. Assay the unfractionated serum, the unbound fraction from the peptide column, and the purified IgG by ELISA using peptide-coated plates, to estimate recovery of peptide-specific IgG. For a good antiserum, yields of IgG will typically be l-2 mg/mL serum. 4. Notes B 1. In order to minimize steric hindrance to antibody binding during affmity chromatography, it is advantageous to immobilize a peptide by the same unique site that was used for attachment to a carrier protein, This site is usually chosen to be as far as possible from the epitope of interest.
Many different carrier proteins have been used successfully for the production of anttpeptide antibodies. Keyhole hmpet hemocyanin usually works well, but occasionally the protein comes out of solution during conjugation by the procedure described. In this event, ovalbumin is a suttable alternative. Since antibodies will also usually be produced against the carrier itself, bovine serum albumin, which is often used as a blocking agent in immunoassays, is not employed. If high-titer antisera are not obtained using these carriers, an alternative is purified protein derivative of tuberculin (PPD), a protein isolated from the culture supematant of Mycobacterium tuberculosis.