Catalog Number: 154010, 154011, 154012, 154013, 154014, 154015, 678001, 678002, 678011, 678012, 678021, 688022
Gold Colloids - 5, 10, 15, 20, 30, and 40 nm


Description: Colloidal gold is an electron-dense, non-fading marker useful as a probe in electron microscopy, light microscopy and immunoblotting.3 It requires no additional processing for detection, but in some applications the signal can be enhanced by reaction with silver.5,6 It can be complexed with biomolecules by strong, non-covalent interactions.8

Choosing a Particle Size: Generally, particles less than 15 nm are most useful in transmission electron microscopy and for applications where access to the probe may be hindered by larger particles.11 Particles greater than 15 nm are more suitable for scanning electron microscopy, light microscopy and blotting.10

Preparation of Antibody/Protein - Gold Conjugates


Materials

All glassware must be scrupulously clean. Glass and plastic containers and stirrers should be cleaned in aqua regia, thoroughly washed in deionized water, and siliconised.

All reagents must be of high quality Analar grade and should be filtered immediately before use. Water should be double distilled or high quality deionized.

Antibody

The antibody should be affinity purified and of the highest quality. On the day of preparation make up a 0.1 ug/ul solution of antibody in 2 mM borax and dialyse for at least 4 hours in 1 liter of borax at pH 9.0. Centrifuge the antibody at 100,000 g for 1 hour at +4C just before use. Keep at +4C.

Buffers

Buffer A: Tris/HCl buffered saline at pH 8.2 with 1% bovine serum albumin (BSA) and 0.1% sodium azide.

Conjugation

Conjugation of antibodies to gold particles depends upon three separate but dependent phenomena: (a) ionic attraction between the negatively charged gold and the positively charged protein; (b) hydrophobic attraction between the antibody and the gold surface; (c) dative binding between the gold conducting electrons and sulphur atoms which may occur within amino acids of the protein.

In order to form a strong absorption between gold and antibody a preliminary titration must be performed to determine the optimum conditions for conjugation.

Preliminary titration
  1. Adjust the gold colloid to pH 9.0. Pipette 1 ml of colloid into each of a series of 3 ml clean plastic tubes.
  2. Adjust the antibody (0.1 ug/ul) to pH 9.2 with 100 mM K2CO3 or 100 mM HCl.
  3. Add the antibody to each tube in a series from 0 - 150 ul (i.e. 0 - 15 ug in steps of 0, 1, 2, 3 ... 15 ug).
  4. Make up each tube to 1.15 ml with 2 mM borax.
  5. Shake each tube and leave for approximately 5 minutes to conjugate.
  6. To each tube add 100 ul of 10% NaCl and agitate for 1 minute.
  7. The tube containing the minimum amount of protein required to stabilize the gold solution is indicated by the one in which the color of the gold solution does not change from red to blue upon the addition of NaCl.

Clusters/pH conjugate

In addition to the titration for judging the minimum amount of protein is also necessary to determine the correct pH for the conjugation. This is best performed at, or near to the isoelectric point of the protein. It is found by performing the preliminary titration at different pH values (eg pH 7, 8, 9, 10) and examining the conjugate (without added salt) in the electron microscope. A spread of the gold particles with absorbed antibody is made by floating a Formvar coated nickel EM grid on a droplet of the conjugate for 30 minutes and then washing in deionized water. The suspension on the grid is observed without further treatment in the EM. The correct pH for conjugation is that in which the conjugation of protein to gold does not produce clusters in the EM.

Final conjugation

Having determined the minimum amount of protein to stabilize the gold solution the amounts may be scaled up. Typical volumes are described here.
  1. Take 100 ml of gold solution and adjust to pH 9.0.
  2. Adjust the dialysed and centrifuged antibody solution (0.1 ug/ul) to pH 9.2.
  3. Add the determined amount of antibody solution dropwise to the gold while stirring rapidly.
  4. After 5 minutes add 10 ml of filtered 10% BSA at pH 9.0 and stir gently for 10 minutes.

Purification

The gold conjugate must be purified from excess antibody and any small clusters removed before concentrating and storing.

1. Spin the gold conjugate at the following speeds according to gold particle size:

2. The gold conjugate will form a loose precipitate at the bottom of the tube. Discard the clear supernatant and resuspend the pellet into 2 ml of buffer A.

3. Prepare a 10 - 30% glycerol gradient (10 ml) in buffer A and carefully load the suspended conjugate onto the top.

4. Spin the gradient in a swing out rotor as follows according to particle size:

5. Carefully pipette out each fraction in 2 ml fractions from the tube and examine in the EM as above. The clustered fractions will be found in the lower fractions.

6. Test each fraction for sensitivity by immunoblotting against the specific antigen blotted onto nitrocellulose or on serially cut tissue sections.

7. Pool those fractions which are acceptable and suspend in buffer A to a dilution having an optical density of 3.0 at 530 nm. Alternatively dilute in buffer A to make a calculated protein concentration of approximately 30 ug/ml from the original volume.

Storage

The gold conjugate, if correctly made, will be stable at +4C for several months. If long term storage is required it should then be aliquoted into 1 ml volumes and 20% glycerol added. It may then be frozen and kept at -20C for years.

Availability:

Catalog NumberDescriptionSize
154010Colloidal Gold Particles, 5 nm1 ml
5 ml
678001
678002
Gold Colloid, 5 nm50 ml
100 ml
154011Colloidal Gold Particles, 10 nm1 ml
5 ml
678011
678012
Gold Colloid, 10 nm50 ml
100 ml
154012Colloidal Gold Particles, 15 nm1 ml
5 ml
678021
678022
Gold Colloid, 20 nm50 ml
100 ml
154014Colloidal Gold Particles, 30 nm1 ml
5 ml
154015Colloidal Gold Particles, 40 nm1 ml
5 ml
980661
980662
Gold Label Diluent, Ready-to-Use diluent for gold labeled antibodies fromulated to maximize stability and minimize background. Subjected to 0.2 micron filtration. Diluted reagents can be stored between 4-8C for up to 18 months.125 ml
500 ml

References:
  1. Ackerman, G.A.,et al., J. Histochem. Cytochem., v. 31, 433 (1983).
  2. Beesley, J. (1989): Colloidal Gold: A new perspective for cytochemical marking. Royal Microscopical Society Handbook, No. 17, Oxford Science Publications. Oxford University Press. MP Catalog # 194224. (This book covers the techniques of gold probe production and immunolabeling. Each technique is described in protocol)
  3. Beesley, J.E., Proc. Royal Micr. Soc., v. 20, 187 (1985).
  4. Birrell, G.B., et al., J. Histochem. Cytochem., v. 35, 843 (1987).
  5. Brada, D. and Roth, J., Anal. Biochem., v. 142, 79 (1984).
  6. Danscher, G. and Norgaard, J.D., Histochem. Cytochem., v. 31, 1394 (1983).
  7. Geoghagen, W.D. and Ackerman, G.A., J. Histochem. Cytochem., v. 24, 1187 (1977).
  8. Geoghegan, W.D., et al., Immunol. Comm., v. 7, 1 (1978).
  9. Roth, J., Techniques in Immunocytochemistry, v. 2, G.R. Bullock and P. Petrusz (eds.), Academic Press: New York, pp. 217-284 (1983).
  10. Slot, J.W. and Geuze, H.J., J. Cell. Biol., v. 90, 533 (1981).
  11. Tolson, N.D., et al., J. Microsc., v. 123, 215 (1981).
  12. Wang, B.L., et al., Histochem., v. 83, 109 (1985).