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"Naked Gold in a Box"
is an easy-to-use kit for preparing highly reactive gold conjugates. The Gold-in-a-Box kit enables you to determine
(in under one hour) the optimal colloidal gold binding conditions for attaching soluble proteins, including the Fc region of purified
monoclonal and polyclonal antibodies. The resulting gold particles are highly reactive and greatly increase the sensitivity of your test.
The Gold-in-a-Box kit helps you determine the ideal pH for coating your protein onto the Naked Gold. Gold colloids which bind ligands through
a sulphur bond have proved highly successful for this application. For optimal binding of the antibody or protein while retaining a high
degree of specific activity, the pH of the gold colloid must be adjusted to slightly above the iso-electric point of the coating protein.
This is done by means of pH titration with the buffers provided. Different amounts of buffers A and B, or C and D, are added to the gold
to create a range of pH values from 5 to 11. The antibody or protein is added and the reaction stopped after 30 minutes.
Kit Components for NGIB18-1
- Naked Gold Sol 20 nm - 2 x 9.0 ml (cap with black ring))
- Buffer Solution A - 1.0 ml (cap with black dot)
- Buffer Solution B - 1.0 ml (cap with green dot)
- Buffer Solution C - 1.0 ml (cap with blue dot)
- Buffer Solution D - 1.0 ml (cap with red dot)
- BSA Blocking Stabilizer Solution - 2.0 ml (clear cap)
- Gold Drying Buffer - 2.0 ml (cap with purple dot)
Kit Components for NGIB18-2
- Naked Gold Sol 40 nm - 2 x 9.0 ml (cap with red ring)
- Buffer Solution A - 1.0 ml (cap with black dot)
- Buffer Solution B - 1.0 ml (cap with green dot)
- Buffer Solution C - 1.0 ml (cap with blue dot)
- Buffer Solution D - 1.0 ml (cap with red dot)
- BSA Blocking Stabilizer Solution - 2.0 ml (clear cap)
- Gold Drying Buffer - 2.0 ml (cap with purple dot)
Kit Components for NGIB18-3
- Naked Gold Sol 40 nm - 9.0 ml (cap with black ring)
- Naked Gold Sol 20 nm - 9.0 ml (cap with red ring)
- Buffer Solution A - 1.0 ml (cap with black dot)
- Buffer Solution B - 1.0 ml (cap with green dot)
- Buffer Solution C - 1.0 ml (cap with blue dot)
- Buffer Solution D - 1.0 ml (cap with red dot)
- BSA Blocking Stabilizer Solution - 2.0 ml (clear cap)
- Gold Drying Buffer - 2.0 ml (cap with purple dot)
Kit Components for NGIB44-1
- Naked Gold Sol 20 nm - 44.0 ml (cap with red ring)
- Buffer Solution A - 2.0 ml (cap with black dot)
- Buffer Solution B - 2.0 ml (cap with green dot)
- Buffer Solution C - 2.0 ml (cap with blue dot)
- Buffer Solution D - 2.0 ml (cap with red dot)
- BSA Blocking Stabilizer Solution - 4.0 ml (clear cap)
- Gold Drying Buffer - 4.0 ml (cap with purple dot)
Kit Components for NGIB44-2
- Naked Gold Sol 40 nm - 44.0 ml (cap with black ring)
- Buffer Solution A - 2.0 ml (cap with black dot)
- Buffer Solution B - 2.0 ml (cap with green dot)
- Buffer Solution C - 2.0 ml (cap with blue dot)
- Buffer Solution D - 2.0 ml (cap with red dot)
- BSA Blocking Stabilizer Solution - 4.0 ml (clear cap)
- Gold Drying Buffer - 4.0 ml (cap with purple dot)
Materials required but not provided: Clean glass or polystyrene test tubes (12 x 75 mm)
Pipettes and tips
Protocols
(Click here for a printable version of the protocol)
Sample preparation
The antibodies or proteins used with this kit must be at a concentration of 1 mg/ml or greater and from 1 mg/ml to 2 mg/ml should be
in half-strength conventional PBS. If they are not, then dialyze the antibody or protein against PBS diluted 1/2. Proteins at a
concentration of 2 mg/ml or greater should be in full-strength PBS.
Generic Procedure
Note: Use aseptic technique when handling the gold colloid.
1. Shake or swirl the colloid to resuspend any settled gold. Place 0.5 ml Naked Gold colloid into ten (10) clean individual test tubes.
2. Label each tube with the pH value (or, 1 through 10) from the provided pH charts.
3. Use the pH charts to add the appropriate amounts of buffer in µl to each test tube. Shake to mix.
pH charts for optimal coating at pH 5-11 (per 0.5 ml of gold)
Tube # pH Buffer A
Buffer B Buffer C
Buffer D
1 5.4 9 µl 1 µl
2 6.6 8 µl 2 µl
3 7.3 6 µl 4 µl
4 7.8 4 µl 6 µl
5 8.2 2 µl 8 µl
6 8.4 10 µl 0 µl
7 8.8 8 µl 2 µl
8 9.2 6 µl 4 µl
9 9.6 4 µl 6 µl
10 10.1 2 µl 8 µl
4. Place each tube on a low speed vortex mixer and add antibody solution (See Sample Preparation Section). Mix thoroughly
(about 2 to 3 seconds). Ideally, for the 40 nm gold colloid, 7 µl of a 2 mg/ml solution of antibody or protein are optimal.
For the 20 nm gold colloid, ideally 14 µl of a 2 mg/ml solution of antibody or protein are optimal.
Note: The saturation point for 40 nm gold colloid is typically near 30 µg of antibody per ml of colloid.
The saturation point for 20 nm gold is about 60 to 70 µg of antibody per ml of colloid.
5. A deepening purple color and/or black precipitate in some tubes indicates that the antibody or protein is below its iso-electric point,
leading to cross-linking of individual gold particles. Cross-linked or deep purple colloids are not useful in immunological assays and
should be discarded. Only tubes with a slight purple color or no change in colour should be used.
6. Allow the reaction to continue for a total of 30 minutes.
Note: See section below on "Stability of Gold Conjugates".
7. Stop the reaction by the addition of 50 µl of Blocking Stabilizer Solution.
Note: In some conjugates that might otherwise give rise to non-specific reactivity, it is often best to allow
the blocker to react for an additional 16 hours at room temperature.
Stability of Gold Conjugates
Gold particles completely coated with protein take on the properties of the coating proteins and become very stable in solutions of high
ionic strength. An excellent way of testing the effectiveness of the coating reaction is to combine 10 µl of coated gold colloid
(prior to the addition of the BSA Blocking Stabilizer Solution) with 10 µl of 1 M NaCl. Incompletely coated colloid will fall
out of solution (turn black), while completely coated colloids will remain stable (red).
Testing of BSA Blocked Gold Conjugates
The gold conjugate is now ready for use in a rapid assay at a nominal amount of 5-15 µl per assay. Tubes showing a slight colour change and
ones with no colour change should be assayed for activity. Tubes with the best activity are usually a good indicator of the
approximate iso-electric point of the coating antibody or protein.
To stabilize the gold conjugate during drying, add 0.1 ml of Gold Drying Buffer for every 1.0 ml of conjugate. Mix thoroughly.
Note: This generic procedure may be modified or scaled as needed. When developing a new assay, it is important to determine the
optimal amount of ligand to add to the gold particles. Once the optimal coating pH has been determined, it is useful to optimize binding
by decreasing or increasing the amount of antibody added to each tube. A 20% increase or decrease in antibody or protein
added often gives an improved coating. In a few cases, a 40% or more increase or decrease in the coating protein concentration may be necessary.
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