rev: March 16, 2005

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SAMPLE  GOLD CONJUGATION PROTOCOL


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 siliconized. All reagents must be of high quality Analytical 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 0.1ug/ul solution of antibody in 2mM borax and dialyze for at least 4 hours in 1 liter of borax at pH9. Centrifuge the antibody at 100,000g for 1h at 4 DEG C just before use. Keep at 4C.

Gold colloid:

Procedures for the preparation of gold colloids of various particle sizes are published elsewhere (1). A wide range of high quality gold colloids ready to use for conjugation to proteins is available. For example given here it is assumed that the gold colloid is prepared from a 0.01% gold chloride solution.

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 pH9. Pipette 1ml of colloid into each of a series of 3ml clean plastic tubes.

2. Adjust the antibody (0.1ug/ul) to pH9.2 with 100nM K2CO3 or 100mM HC1.

3. Add the antibody to each tube in a series from 0-150ul (ie 0-15ug in steps of 0, 1, 2, 3 ....15ug).

4. Make up each tube to 1.15ml with 2mM borax.

5. Shake each tube and leave for approximately 5 minutes to conjugate.

6. To each tube add 100ul of 10% NaC1 and agitate for 1 minute.

7. The Tube containing the minimum amount of protein required to stabilize the gold sol is indicated by the one in which the color of the gold sol does not change from red to blue upon the addition of NaCl.

Clusters/pH of 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 conjugated (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 cluster in the EM.

Final conjugation

Having determined the minimum amount of protein to stabilize the gold sol the amount may be scaled up.Typical volumes are described here.

1. Take 100ml of gold sol and adjust to pH9

2. Adjust the dialyzed and centrifuged antibody solution (0.1ug/ul) to pH9.2

3. Add the determined amount of antibody solution dropwise to the gold while stirring rapidly

4. After 5 minutes add 10ml of filtered 10% BSA at pH9 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:

5nm        45,000g   for 1h @ 4C

10nm      25,000g   for 1h @ 4C

15nm      15,000g   for 1h @ 4C

20nm        8,000g   for 1h @ 4C

40nm        6,000g  for 1 h@ 4C
These speeds and times are approximate and must be determined experimentally.

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

3. Prepare a 10-30% glycerol gradient (10ml ) 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:

5nm 125,000g for  1hour at 4C

10nm 50,000g for  1hour at 4C

15nm 15,000g for  1hour at 4C

20nm 12,000g for 1 hour at 4C

40nm  6,000g for 1 hour 4 DEG C

These speeds and times are approximate and must be determined experimentally.

5. Carefully pipette out each fraction in 2ml 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 a optical density of 3.0 at 520nm. Alternatively dilute in buffer A to make a calculated protein concentration of approximately 30ug/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 is should be aliquoted into 1ml volumes and 20% glycerol added. It may then be frozen and kept at -20C for years.

Reference

1. Beesley J (1989) "Colloidal Gold. A new perspective for cytochemical marking". Royal Microscopical Society Handbook No 17.Oxford Science Publications. Oxford University Press.

(Good for beginners. Gives protocols for making and using gold conjugates.Describes their use in a range of microscopical applications).

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