Worm Breeder's Gazette 8(1): 54
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Use of Colloidal Gold Particles in EM We have been using a simple and inexpensive procedure to produce colloidal gold particles of a uniform size (5 nm) which can be conjugated directly to proteins. Gold particles serve as excellent electron dense markers for use in electron microscopy. Preparation of Colloidal Gold Particles by Ultrasonics 1. Dilute 0.1 ml of 1% gold chloride [ H(AuCl4) ] in 50 ml dH20. 2. Make solution neutral with 0.2M K2CO3. 3. Immediately before sonication, add 0.5 ml of 100% ethanol as a catalyst. 4. Carry out sonication at 20 Kc and 125 W by immersing a flat-end probe approximately 1 cm under the surface of the gold solution. 5. After about 2-5 min, the solution attains a pinkish color with maximum intensity at A = 520 nm. Note: To prevent the solution from heating up during sonication, keep it cool in an ice water bath. This helps prevent the formation of larger gold particles. Store at 4 C. Preparation of Gold Labelled Protein 1. Mix 1mg of purified protein with 5ml of colloidal gold solution and incubate for 15 min at room temperature with mixing. 2. Add 10mg BSA, mix, and incubate for another 5 min to prevent aggregation of gold-protein complexes. 3. Add NaCl (100g/l) to make a final concentration of 10g/l. 4. Centrifuge at 1700 g for 20 min to remove excess BSA. 5. Centrifuge supernatant from step 4 at 60,000 g for 15 min to bring down gold labelled protein, leaving unlabelled protein in the supernatant. 6. Pellet should be a loosely packed, dense red sediment and can be resuspended in 0.5 to 1.0 ml of desired buffer. Use undiluted.