Preparation of Colloidal Gold Particles and Conjugation to Protein A, IgG, F(ab ) 2, and Streptavidin

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1 Chapter 8 Preparation of Colloidal Gold Particles and Conjugation to Protein A, IgG, F(ab ) 2, and Streptavidin Sadaki Yokota Abstract Colloidal gold probes, including protein A-, IgG-F(ab ) 2 -, and streptavidin-labeled gold particles, are useful tools for localization of antigens in cells and tissues by immunoelectron microscopy (IEM). This chapter describes different methods for the preparation of colloidal gold and conjugation of colloidal gold to protein A, IgG, and streptavidin. Key words: Preparation of colloidal gold, sizing of gold particles, protein A-gold, IgG-gold, streptavidin-gold. 1. Introduction Colloidal gold is an electron-dense spherical particle of 2 40 nm in diameter that can be conjugated to proteins and is clearly recognizable in the electron microscope. These properties can be exploited to localize target molecules in thin sections of cells and tissues by immunoelectron microscopy (IEM). IEM uses colloidal gold probes conjugated with antibodies or protein A/G to detect antigens in thin plastic (1) or frozen sections (2). In addition, colloidal gold particles of various sizes can be separately produced to allow labeling of two or more different target molecules in the same section. Finally, gold particles are easily counted in electron microscopic images, enabling accurate and useful quantitative analysis of the labeling (1). Preparing gold probes for IEM essentially involves two steps: preparation of colloidal gold particles of specific sizes and S.D. Schwartzbach, T. Osafune (eds.), Immunoelectron Microscopy, Methods in Molecular Biology 657, DOI / _8, Springer Science+Business Media, LLC

2 110 Yokota conjugation of the gold particles to proteins such as protein A/G, IgG, and streptavidin. In the first step, monodispersed colloidal gold is prepared by reduction of tetrachloroauric acid. The size of the colloidal gold particles produced is determined by the reducing reagent used and its concentration (3). In the second step, colloidal gold particles of uniform size are conjugated to primary antibodies with a specific binding affinity for the target molecule or to protein A/G, streptavidin, or a secondary antibody that has a specific affinity for the primary antibody recognizing the target. Although a range of pre-prepared immunogold probes are commercially available, they can be easily produced at low cost in the ordinary cell biology laboratory. This chapter first details the preparation of colloidal gold particles of various sizes, including handling of the glass reaction vessels used, the general properties of colloidal gold particles, and how to check the size of gold particles prepared. Methods for conjugating the colloidal gold particles to proteins, including protein A, IgG, F(ab ) 2, and streptavidin, are then described with a brief review of the properties of these proteins. Finally, the chapter will note methods for titrating the minimum amount of protein needed to stabilize colloidal gold preparations and for removing non-specific proteins from the colloidal gold probes before use. 2. Materials 2.1. Colloidal Gold Preparation 1. Double-distilled water (DDW) is used for all solutions and procedures. 2. 4% gold(iii) chloride trihydrate (Sigma-Aldrich, St. Louis, MO): Prepare a 4% stock solution and store at 4 C(see Note 1). Dilute to final concentration just before use M potassium carbonate: Store at room temperature (RT) M sodium thiocyanate. Harmful irritant. Store at RT M sodium borohydride. Highly flammable, toxic. Store at RT mm and 38.8 mm trisodium citrate: Store at RT. 7. Siliconized flasks (see Note 2). In a hood with gloves and safety glasses, soak the inner surface of the glassware with 5% dimethyldichlorosilane in heptane until the heptane evaporates. Wash the glassware with DDW and use only for colloidal gold preparation. 8. Magnetic stirring plate and stir bars.

3 Colloidal Gold Particles and Conjugation Titration to Determine Minimum Amount of the Protein to be Conjugated Needed to Stabilize the Colloidal Gold 1. 10% NaCl. Store at RT M acetic acid. Store at RT Preparation of Protein A-Colloidal Gold Probes 1. Protein A (GE Healthcare Bio-Sciences Corp. Piscataway, NJ). Store at 20 C. 2. BSA, bovine serum albumin (Sigma-Aldrich, St. Louis, MO). Store at 20 C % glycerol. Store at RT. 4. Phosphate-buffered saline (PBS): Prepare a 10 stock solution by mixing 80 g NaCl, 2 g KCl, 11.5 g Na 2 HPO 4 7H 2 O, 2 g KH 2 PO 4, and DDW for a final concentration of 1 L. Autoclave and store at 4 C. Dilute 10-fold to use Conjugation of IgG or F(ab ) 2 with Colloidal Gold 1. 2 mm borate HCl buffer (ph 9.0). Prepare a 12.5 mm stock solution by mixing 4.6 ml of 0.1 M HCl, 50 ml of M sodium borate, and add DDW for a final volume of 100 ml. Autoclave and store at RT. Dilute 6.25-fold to use. 2. Millipore filters with 0.45-μm pore size (Millipore Corporation, Billerica, MA) mm Tris HCl, ph 8.2. Prepare a 100 ml 10 stock solution of 0.2 M Tris HCl, ph 8.2, autoclave and store at 4 C. Dilute 10-fold to use. 4. Triton X-100 (Sigma-Aldrich, St. Louis, MO). Harmful irritant. Store at RT. 5. Polyethylene glycol 20,000 (Sigma-Aldrich, St. Louis, MO). Store at RT. 6. Polyvinylpyrrolidone (M.W. 10,000) (Sigma-Aldrich, St. Louis, MO). Store at RT ml ultraclear centrifuge tubes (Beckman Coulter, Fullerton, CA). 8. SW50.1 ultracentrifuge rotor (Beckman Coulter, Fullerton, CA). 9. Ultracentrifuge (Beckman Coulter, Fullerton, CA). 10. Affinity-purified IgG (MP Biochemicals, Solon, OH). 11. Affinity-purified F(ab ) 2 (Abcam, Cambridge, MA).

4 112 Yokota 2.5. Conjugation of Streptavidin with Colloidal Gold 1. Streptavidin (Sigma-Aldrich, St. Louis, MO). Store at 20 C M sodium bicarbonate. Store at RT. 3. Polyethylene glycol 6,000 (Sigma-Aldrich, St. Louis, MO). Store at RT. 4. Sodium azide. Highly toxic and corrosive. Hazardous material for the environment. Store at RT. 3. Methods 3.1. Colloidal Gold Preparation Preparation of Colloidal Gold with a Particle Size of 2 3 nm in Diameter Preparation of Colloidal Gold with a Particle Size of 4 5 nm in Diameter Preparation of Colloidal Gold with a Particle Size of 8.5 nm in Diameter 1. Put 50 ml of 1% gold(iii) chloride trihydrate into a clean 200 ml siliconized flask (4). 2. Add0.75mLof0.2MK 2 CO 3 to this flask. 3. Stir the mixture on a magnetic stirrer while quickly adding 0.3 ml of 1 M sodium thiocyanate. 4. Allow the mixture to stand in the dark without stirring for h. The final color of the colloidal gold solution should be yellow (see Note 2). 5. Check the size of the colloidal gold particles with an electron microscope (see Section 3.1.7) 1. Add 0.5 ml of 1% gold(iii) chloride trihydrate solution to 50 ml of DDW in a 100 ml siliconized flask (5). 2. Stir this solution with a magnetic stirrer and quickly add 0.3 ml of 1 M sodium borohydride. 3. The color of the mixture should change immediately to reddish brown then to a dark red wine color within 20 min. 4. Keep the mixture overnight at 4 C. 5. Check the size of the colloidal gold particles with an electron microscope (see Section 3.1.7). 1. Heat 106 ml of 2.2 mm trisodium citrate in a 200 ml flask until boiling (6). 2. Immediately add 1 ml of 24.3 mm gold(iii) chloride trihydrate and stir. 3. Continue to boil the mixture with stirring for 15 min. 4. The color should change to yellowish red during heating 5. Maintain the starting volume of the mixture by addition of DDW as necessary during boiling. 6. Cool the mixture in an ice bath.

5 Colloidal Gold Particles and Conjugation Check the size of the colloidal gold particles with an electron microscope (see Section 3.1.7) Preparation of Colloidal Gold with a Particle Size of 12 nm in Diameter Preparation of Colloidal Gold with a Particle Size of 15 nm in Diameter Preparation of Colloidal Gold with a Particle Size of nm in Diameter (see Note 3) 1. Heat 243 ml of 0.3 mm gold(iii) chloride trihydrate in a clean 400 ml siliconized flask until boiling (6). 2. With vigorous stirring, quickly add 8.5 ml of 38.8 mm trisodium citrate to the boiling solution. 3. Continue to boil for 15 min. 4. Maintain the volume of the mixture with DDW as necessary. 5. The color should change from dark blue to wine red during boiling. Cool the mixture in an ice bath and check the size of the colloidal gold particles with an electron microscope (see Section 3.1.7). 1. Heat 243 ml of 0.3 mm gold(iii) chloride trihydrate in a clean 400 ml siliconized flask until boiling (6). 2. While stirring vigorously, quickly add 7.5 ml of 38.8 mm trisodium citrate. 3. Continue to boil for 15 min. 4. Maintain the volume of the mixture with DDW as necessary. 5. The color should change from blue to wine red during boiling. 6. Cool the mixture in an ice bath and check the size of the colloidal gold particles with an electron microscope (see Section 3.1.7). 1. Heat 50 ml of 0.01% gold(iii) chloride trihydrate solution in a 100 ml flask until boiling (3) 2. While boiling, quickly add the volume ( ml) of 1% trisodium citrate solution (Table 8.1) required to produce the desired size colloidal gold particles. The color of the boiling mixture should turn faint blue within 25 s and then 70 s later suddenly change to brilliant red. 3. Continue to boil for a further 5 min until reduction of the gold chloride is complete as indicated by the solution becoming the color shown in Table Cool the mixture in an ice bath and check the size of the colloidal gold particles with an electron microscope (see Section 3.1.7) Determining the Size of Colloidal Gold Particles 1. Float a Formvar-coated nickel grid on a drop of 10% BSA PBS solution for 10 min to coat the membrane with BSA.

6 114 Yokota Table 8.1 The amount of trisodium citrate to add to 50 ml of 0.01% gold(iii) chloride trihydrate to obtain colloidal gold particles of the specified size a Diameter of colloidal gold particles (nm) Amount (ml) of 1% trisodium citrate Color of colloidal gold solution Orange Red Red Dark red Violet Violet a Modified from Frens (7) 2. Wash the grid with a jet of DDW for several seconds. 3. Float the grid on a drop of the colloidal gold solution for 5 min. The gold particles will bind the BSA coating the membrane. 4. Wash the grid with a jet of DDW for several seconds and air dry. 5. Take electron micrographs at 80,000 magnification and enlarge them photographically or digitally to 400, Measure the diameter of the gold particles and calculate the actual size of the colloidal gold particles. Electron microscopic images of colloidal gold particles of different sizes are shown in Fig Fig Electron microscopic images of gold particles. (A) 2 3-nm gold particles. (B) 4 5-nm gold particles. (C)8.5-nm gold particles. (D) 15-nm gold particles. (E) 28-nm gold particles. Bar = 50 nm.

7 Colloidal Gold Particles and Conjugation Titration to Determine Minimum Amount of the Protein to be Conjugated Needed to Stabilize the Colloidal Gold 3.3. Preparation of Protein A-Colloidal Gold Probes Conjugation of Protein A to Colloidal Gold (see Notes 5 and 6) 1. Pipette 100 μl of DDW containing 0, 0.5, 1, 2, 3, or 4 μg of the protein to be conjugated into six test tubes, respectively. 2. Adjust the ph of the colloidal gold solution to ph 5.2 using 0.1 M acetic acid. Measure the ph by pipetting a drop of the solution (2 3 μl) onto a piece of litmus paper (see Note 4). 3. Into each tube, pipette 0.5 ml of colloidal gold solution. After 10 min at RT, pipette 0.5 ml of 10% NaCl into each tube with agitation. 4. The tube containing the minimum amount of protein to be conjugated required to stabilize 0.5 ml colloidal gold solution is the tube containing the least protein and no bluish black precipitate after the addition of NaCl. 1. Place 20 ml of the colloidal gold solution you prepared into a beaker and adjust the ph to 5.2 using 0.1 M acetic acid. Measure the ph by pipetting a drop of the solution (2 3 μl) onto a piece of litmus paper. 2. Prepare a solution of 1 mg/ml protein A in distilled water. 3. Based on the least amount of protein A needed to stabilize 0.5 ml colloidal gold (determined as described in Section 3.2), pipette 10% more than this amount of protein A into the beaker containing 20 ml colloidal gold solution you prepared (see Note 7). 4. Allow to stand for 10 min. 5. Add 1 ml of 10% BSA to the gold solution to further stabilize the gold conjugate. 6. Using a fixed angle rotor, centrifuge the gold-protein A conjugate at the following settings for 30 min: 15,000 g for 15-nm gold conjugate and larger, 35,000 g for 8-nm gold conjugate, and 100,000 g for 2 3-nm gold conjugate. Aggregated protein A-gold conjugate forms a tight pellet on the side of the tube while the non-aggregated conjugate pools loosely on the bottom of the tube. 7. Slowly remove the supernatant from the centrifuge tube using a capillary tube connected to an aspirator. Remove as much of the non-bound protein A containing supernatant as you can being careful to avoid disturbing the conjugated protein A pooled at the bottom of the tube. 8. Collect the dark red mass of gold-protein A conjugate pooled at the bottom of tube by resuspending it in 50% glycerin PBS (see Note 8).

8 116 Yokota Removal of Non-conjugated Protein A (see Note 9) 3.4. Conjugation of Affinity-Purified IgG and F(ab ) 2 with Colloidal Gold 1. In a 5 ml ultraclear centrifuge tube, overlayer a 1 ml 50% glycerol PBS cushion with 3.5 ml of 10% glycerol PBS. 2. Layer 0.4 ml of the non-aggregated protein A colloidal gold conjugate onto the top of the tube. 3. Centrifuge in a Beckman SW50.1 rotor for 30 min at 15,000 g for 15-nm gold conjugate and larger, 35,000 g for 8-nm gold conjugate, and 100,000 g for 2 3-nm gold conjugate. 4. Using an aspirator, remove the supernatant leaving the 50% glycerol cushion containing the colloidal gold-conjugated protein A. Collect the colloidal gold-conjugated protein A and mix it with 50% glycerol-pbs. 5. Add 10% BSA to the protein A-gold conjugate to a final concentration of 0.5%. 6. Store the protein A-gold conjugate at 20 C until use (see Note 9). 1. Dialyze IgG or F(ab ) 2 solution (1 mg/ml) against 2 mm borate HCl buffer (ph 9.0). Remove precipitates by centrifugation at 10,000 g for 1 h (7, 8). 2. Place 1 ml of the colloidal gold solution you prepared into a beaker. Adjust the ph to 9.0 with 0.2 M K 2 CO 3 just before use. 3. Based on the least amount of IgG or F(ab ) 2 needed to stabilize 0.5 ml colloidal gold (determined as described in Section 3.2), pipette 10% more than this amount of IgG or F(ab ) 2 into the beaker containing 1 ml colloidal gold solution you prepared (see Note 7). Allow to stand for 10 min. 4. Add BSA to the mixture to a concentration of 1% BSA. 5. Filter the mixture through a 0.45-μm Millipore filter. 6. Using a fixed angle rotor, centrifuge the gold IgG or gold F(ab ) 2 conjugate at the following settings for 30 min: 15,000 g for 15-nm gold conjugate and larger, 35,000 g for 8-nm gold conjugate, and 100,000 g for 2 3-nm gold conjugate. Aggregated IgG and F(ab ) 2 gold conjugates form a tight pellet on the side of the tube while the nonaggregated conjugates pools loosely on the bottom of the tube. 7. Slowly remove the supernatant from the centrifuge tube using a capillary tube connected to an aspirator. Remove as much of the non-bound IgG or F(ab ) 2 containing supernatant as you can, being careful to avoid disturbing the conjugated IgG or F(ab ) 2 pooled at the bottom of the tube (see Note 11).

9 Colloidal Gold Particles and Conjugation Collect non-aggregated conjugate at the bottom of the tube, mix with 0.5 ml 20 mm Tris HCl buffer (ph 8.2) containing 1% BSA, and re-centrifuge as before. Repeat twice. 9. Mix conjugate with a small amount of 20 mm Tris HCl (ph 8.2), 40% glycerol, 1% BSA, 0.01% Triton X-100. Store the conjugate at 20 C(see Note 12) Conjugation of Streptavidin with Colloidal Gold 1. Dissolve 500 μg streptavidin in 0.5 ml of 1 mm phosphate buffer (ph 7.4) (9). 2. Add 200 μl of1mnahco 3 and the streptavidin solution to 20 ml of the colloidal gold solution you prepared. 3. After 10 min of stirring, add 200 μl of 2% polyethylene glycol 6, Using a fixed angle rotor, centrifuge the gold streptavidin conjugate at the following settings for 30 min: 15,000 g for 15-nm gold conjugate and larger, 35,000 g for 8-nm gold conjugate, and 100,000 g for 2 3-nm gold conjugate. Aggregated streptavidin-gold conjugate forms a tight pellet on the side of the tube while the non-aggregated conjugate pools loosely on the bottom of the tube. 5. Slowly remove the supernatant from the centrifuge tube using a capillary tube connected to an aspirator. Remove as much of the non-bound streptavidin containing supernatant as you can being careful to avoid disturbing the conjugated streptavidin pooled at the bottom of the tube. 6. Suspend the non-aggregated conjugate at the bottom of the tube in 0.1 M phosphate buffer (ph 7.4), 0.02% polyethylene glycol 6,000, 0.05% NaN 3.Storeat4 C. 4. Notes 1. The concentration of gold(iii) chloride trihydrate solutions can be measured by absorption at 290 nm. The absorption coefficient for a 0.3 mm gold(iii) chloride trihydrate solution is ± Using different reducing agents on gold(iii) chloride trihydrate produces varying sizes of gold colloid. 2. Aggregation and precipitation of the colloidal gold during preparation can be avoided by siliconizing the glassware used. The color change of the colloidal gold solution must be carefully watched during the reaction. When the reduction is completed, the color of the solution is the color indicated in the text.

10 118 Yokota 3. Gold particles with a diameter >30 nm are unstable even after protein conjugation and therefore should not be stored for long periods. In addition, large-size gold probes are dissociated relatively easily from sections during the IEM procedure (10). 4. Standard electrodes may be damaged by colloidal gold; therefore litmus paper is used for measuring the ph of colloidal gold solutions. Colloidal gold of smaller particle size requires more protein to stabilize it than those preparations containing larger colloids. 5. Colloidal gold is a negatively charged suspension and its stability is maintained by static repelling forces. Therefore, the suspension is unstable and precipitates immediately in the presence of electrolyte. However, when macromolecules such as proteins are absorbed onto the surface of colloidal gold particles by electrostatic van-der- Waals forces, a stable complex is formed that is no longer precipitated in solutions containing electrolytes. Proteins absorbed by gold particles do not lose their enzymatic activity or ligand affinity making them useful for detecting enzyme substrate reactions and receptor ligand interactions. 6. Protein G and protein L are conjugated to colloidal gold by the same method. 7. For example, if the minimum amount of protein A needed is 2 μg/0.5 ml colloidal gold, 80 μg of protein A is needed to saturate 20 ml of colloidal gold. The 10% extra thus requires a total of 88 μg. 8. The color of the colloidal gold conjugate prepared using thiocyanate is dark yellow. 9. When the protein A-gold probes are contaminated with even small amounts of free protein A, the binding of protein A-gold probes to IgG is greatly decreased due to the binding of free protein A to IgG. The most effective way to remove free protein A is the discontinuous glycerol gradient centrifugation method. Repeating the differential centrifugation twice can also be used to eliminate free protein A. 10. The protein A-gold conjugates retain its biological activity for 5 years under these storage conditions. 11. After centrifugation, excess medium attached to the tube wall is wiped off with tissue paper. This almost completely avoids contamination with free IgG or F(ab ) 2.

11 Colloidal Gold Particles and Conjugation Under these storage conditions, the conjugates retain binding activity for 1 year. During storage, the conjugates slowly precipitate and should be centrifuged to remove the aggregated conjugates immediately before use. References 1. Roth, J. (1982) The protein A-gold (pag) technique A qualitative and quantitative approach for antigen localization on thin sections. In Techniques in Immunocytochemistry, Bullock, G. R. and Petrusz, P., eds. Academic Press, London, UK, vol. 1, Tokuyasu, K. T. (1980) Immunochemistry on ultrathin frozen sections. Histochem. J. 12, Frens, G. (1973) Controlled nucleation for the regulation of particle size in monodisperse gold suspensions. Nat. Phys. Sci. 241, Baschong, W., Lucocq, J. M., and Roth, J. (1985) Thiocyanate gold : small (2 3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy. Histochemistry 83, Tschopp, J., Podack, E. R., and Müller- Eberhard, H. J. (1982) Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9- polymerizing complex C5b 8. Proc. Natl. Acad. Sci. USA 79, De Roe, C., Courtoy, P. J., and Baudhuin, P. (1987) A model of protein-colloidal gold interaction. J. Histochem. Cytochem. 35, De Mey, J., Moeremans, M., Geuens, G., Nuydens, R., and De Brabander, M. (1981) High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method. Cell Biol. Int. Rep. 5, Ackerman, G. A., Wolken, K. W., and Gelder, F. B. (1980) Surface distribution of monosialoganglioside Gm1 on human blood cells and the effect of exogenous GM1 and neuraminidase on cholera toxin surface labeling. A quantitative immunocytochemical study. J. Histochem. Cytochem.28, Bonnard, C., Papermaster, D. S., and Kraehenbuhl, J.-P. (1984) The streptavidin-biotin bridge technique: application in light and electron microscope immunocytochemistry. In Immunolabeling for Electron Microscopy, Polak, J. M. and Varndell, I. M., eds. Elsevier, Amsterdam, The Netherlands, Yokota, S. (1988) Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry. J. Histochem. Cytochem. 36,

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