By Graham J.M., Higgins J.A.

This moment quantity of protocols deals the main entire set of contemporary analytical strategies to be had for learning the structure and serve as of membranes. It positive aspects the applying of biochemical, spectroscopic, and fluorimetric the right way to the research of molecular topology, the dynamic points of membrane constitution, and ion delivery. Antibody expertise, research of molecules eager about intracellular signaling, and receptor-ligand interactions also are lined.

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1. Peptide-Carrier Coqjugation The following method, adapted from the procedure described by LaRochelle et al. (3), is designed for the attachment of cysteine-containing peptides to carrier proteins (see Note A3). 1. 4. Dialyze overnight at 4°C against 2 L of this buffer then briefly centrifuge to remove any msoluble material. 2. To 500 pL of the supematant (8 mg protein) add 10 pL 50 mMZV-ethylmaleimide and incubate for 10 mm at 25OC to block free -SH groups on the carrier. 3. Slowly add 200 pL of Sulfo-MBS solution (seeNote A3) and incubate at 25OC for 30 min.

The same procedure as that described for coating with peptides can be used, except that a carrier protein concentration of up to 5 pg/mL should be used. In order to detect antibodies that recognize a peptide sequence in the intact protein, identical condtttons can be used to coat wells with a suspension of membranes contaming the protein of interest. A typical result is shown in Fig. 1, where antisera raised agamst peptides comprising residues 23 l246 and 34-60 of the human erythrocyte glucose transporter (2) were incubated with microtiter plates coated with the purified, membrane-embedded transport protein.

And Singer, S J. (1985) Anomalous interaction of the acetyl choline receptor protein with the nonionic detergent Triton X-114 Proc. Nut1 Acad. Sci. USA 82,958-962 5. Volk, T. and Geiger, B. (1986) A-CAM: A 135kD receptor of intercellular adherens junctions. I. Immunoelectron macroscopic localization and biochemical studies. J. Cell Biol. 103, 1441-1450. 6. Muller, W. A and Gimbrone, M. , Jr. (1986) Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity. analysis by surface-selective iodmation.

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