By Graham J.M., Higgins J.A.
This moment quantity of protocols bargains the main accomplished set of contemporary analytical options on hand for learning the structure and serve as of membranes. It positive aspects the applying of biochemical, spectroscopic, and fluorimetric how to the research of molecular topology, the dynamic elements of membrane constitution, and ion delivery. Antibody know-how, research of molecules desirous about intracellular signaling, and receptor-ligand interactions also are lined.
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2. 1. Preparation of the Column 1. 5. 2. Dithiothreitol (DTT) solution (OSM). This solution can be kept for l-2 mo at -2OOC. 3. SephadexG-10 preswollen in the Tris buffer and suitable column. 4. SulfolinkTM coupling gel (seeNote Bl). 5. 5. This solution should be madeup freshly and usedimmediately. 6. 1MNaCI. 7. 2. 8. 5. 9. 5,5’-Dithiobis (Znitrobenzolc acid) solution (DTNB, 10 mM). 5 mg NaHC03 in 5 mL of the phosphatebuffer. Store at -20°C. 10. Oxygen-free nitrogen. 11. Screw-cappedglass tubes (10 mL).
5. 5. Mix gently, centrifuge, and then remove the supernatant. Repeat this process another three times. 6. To block excess iodoacetyl groups on the gel, incubate for 1 h at 25°C with 5 mL cysteine solution, mixing by gentle inversion. 7. Remove noncovalently bound peptide by transferring the gel back to the filter funnel then washing with 50 mL 1M NaCl. 54 Baldwin 8. Equilibrate the gel in PBS by washing with 50 mL of this buffer. Then pack mto a 5-n& chromatography column. 02% (w/v) sodium azide as a preservative.
Dithiothreitol (DTT) solution (OSM). This solution can be kept for l-2 mo at -2OOC. 3. SephadexG-10 preswollen in the Tris buffer and suitable column. 4. SulfolinkTM coupling gel (seeNote Bl). 5. 5. This solution should be madeup freshly and usedimmediately. 6. 1MNaCI. 7. 2. 8. 5. 9. 5,5’-Dithiobis (Znitrobenzolc acid) solution (DTNB, 10 mM). 5 mg NaHC03 in 5 mL of the phosphatebuffer. Store at -20°C. 10. Oxygen-free nitrogen. 11. Screw-cappedglass tubes (10 mL). 12. Bench-top centrifuge. 2. Affinity-Purification 53 of Antibody 1.