Gold Conjugates

Gold Conjugates, a superior gold probe for light and electron microscopy.

• Probe Stability - Long term stability when stored at -25°C or less.
• Clustering - 85% singlets with no clusters greater than triplets
• Concentration and Dilution - EM and LM probes can be diluted from 1:10 to 1:200. Blotting probes can be diluted from 1:100 to 1:500
• Size Distribution - EM grade probes have a low coefficient of variation, making them quite suitable for multiple labelling applications
• Particle Sizes - Unconjugated colloidal gold sols come in a wide range of sizes: 2, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200 and 250nm

Super Ice® Service, when necessary for shipment of heat labile products (e.g. gold conjugates, resins and some fixatives)

Gold particle size will influence labelling density and selection of instrument magnification when used in immunoelectron microscopy. The tables and figures below should aid the researcher in choosing the gold particle best suited to his or her research requirements (reproduced with permission from Giberson and Demaree: The Influence of Immunogold Particle Size on Labelling Density. Microscopy Research and Technique 27:355-357, 1994).

Table 1: Gold Particle Labelling Densities

Table 2: Percent Decrease in Labelling Density for Paired Comparisons of Gold Conjugates

1 mean diameters 5.5, 10.0, 14.6, 20.4, 28.4
2 mean diameters not given, reported as 5, 15, 30 and 40nm
3 mean diameters 4.92, 10.1, 15.9nm
4 mean diameters not given, reported as 5 and 10nm
5 mean diameters 6.7 and 12.1
6 mean diameters not given, reported as 5, 12, 18, 28, and 38nm
# data extrapolated from graphs and figures to determine percentage decreases
* paired comparisons not statistically significant

Table 3: Paired Comparisons of Gold Particle Size Densities

+ = Significantly Different at 0.05% Level
0 = Not Significantly Different

Labeling of the characteristic electron dense granules found in the processed cheese sample is clearly evident for 5 different sized gold conjugates.

The magnification for each is identical (60,000).

A: 5nm, B: 10nm, C: 15nm, D: 20nm, E: 30nm, Bar in A-E= 0.5µm

Gold Conjugate Technical Information

Notes:

Gold conjugates 5nm to 30nm are packaged with 20% glycerol and can be frozen. 1nm conjugates are not packaged with glycerol and should not be frozen. EM Gold conjugates - %CV: <12% for 5nm; <8% for 10,15 & 20nm; <20% for 30nm.

%Singlets: >85% for all conjugates except Protein A >95%.

Guidelines for use

The gold conjugates are made to the highest specification and will yield excellent results when correctly used. The guidelines discussed herein are designed to help achieve optimum results for post-embedding immunolabeling (labeling of ultrathin or semithin sections) for lelectron or light microscopy. Gold conjugates are supplied in one of the two buffers listed below. The type of buffer will determine the shelf life and storage conditions of the conjugate.

Buffer A: 20mM Tris (tris-hydroxymethyl-aminomethane); 20mM sodium azide; 225mM NaCl; 1% BSA; 20% glycerol. pH 8.2

Conjugates supplied in the buffer: all EM (electron microscopy), LM (light microscopy) and BL (blotting) grade conjugates.

Shelf life and storage conditions: Stable for 12 months at 4°C or for years if frozen at ­25°C or below.

Buffer B: 10mM Na2HPO4; 10mM sodium azide; 150mM NaCl; 1% BSA. pH 7.4
Conjugates supplied in the buffer: All 1nm (ultra small) gold conjugates

Shelf life and storage conditions: Stable for 12 months at 4°C - DO NOT FREEZE

The following suggestions have given good to excellent immunolabeling results at the EM level. For excellent discussion on immunolabeling strategies we recommend: Griffiths, G. (1993) Fine Structure Immunocytochemistry. Springer-Verlag, New York (Although there are a number of buffers which can be used for immunolabeling we list only two:

1. Tris Buffer: 0.242g (20mM) Tris (tris-hydroxymethyl-aminomethane) 0.9g (225mM) NaCl ultrapure water to make 100ml. Adjust pH from 7.4 to 8.2 with 1N NaOH.
2. Phosphate Buffer: 0.148g Na2HPO4 = 0.043g KH2PO4 = 0.72g NaCl ultrapure water to make 100ml. Adjust pH from 7.4 to 8.2 with 1N NaOH.

Positive/Negative Controls:

The use of a positive control is important for working out dilutions of both primary and secondary antibodies and identifying an optimum signal-to-noise ratio. The positive control will also help to establish antigenic locations and what, if any, blocking measures may be required. The use of a negative control is important in the determination of background due to the gold conjugated secondary (omit the primary antibody step and incubate with buffer only), and specificity/background due to the primary antibody (use preimmune sera or an antibody that is nonspecific for the tissue in question).

Fixation:

All antigens are different with respect to fixation. The use of paraformaldehyde alone (2-4%) and paraformaldehyde mixed with glutaraldehyde (0.1-0.5%) is a good place to start. It is possible, with some antigens, to follow with reduced osmium tetroxide (2% aqueous osmium mixed with and equal volume of 3% potassium ferricyanide either, buffered or unbuffered). If osmium can not be used, Berryman and Rodewald (1990, J. Histochem Cytochem 38:159-170) have an excellent protocol to achieve membrane contrast without the use of osmium. Similar results can be achieved by staining the labeled sections for 5 minutes on 2% aqueous uranyl acetate followed by 2% aqueous osmium tetroxide for the same time. We prefer to use 0.1M sodium cacodylate as the buffer at a pH between 7.2-7.4. There are many acceptable buffers and buffer combinations, however, that can be used. (See also Hyat, M.A. (1989) Principles and Techniques of Electron Microscopy: Biological Applications, 3rd Edition, CRC Press, Boca Raton, FL)

Resins:

The approach as to what resin to use is based on immunoreactivity. Resin-free cryosections would head the list followed by hydrophilic polar resins (i.e. Lowicryl K4M, K11M, LR White, LR Gold, Unicryl) which are partially water-soluble, hydrophobic apolar resins (i.e. Lowicryl HM20, HM23) and finally the hydrophobic epoxy resins (i.e. Epon, Araldite, Spurr's).

Sample Labeling Protocol:

1. Blocking Step: 20-30 minutes at room temperature.
2. Primary Antibody Incubation: 30-60 minutes in duration. Can be done at room temperature or 37°C.
3. Buffer Rinse: If done on drops, use at least 6 drops (5 minutes each drop).
4. Gold Conjugate Incubation: 30-45 minutes in duration. Can be done at room temperature or 37°C.
5. Rinse: In the first rinse we have found it beneficial to elevate the NaCl concentration to ~2.5M from 225mM. This appears to aid in eliminating much of the background due to ionic attraction of the negatively charged gold particle. This step is about 10 minutes followed by rinses in distilled water.
6. Post Stain: 2% aqueous uranyl acetate for 5 minutes, rinse well in distilled water followed by 5 minutes with lead stain (25ml distilled water, dissolve one sodium hydroxide pellet then add 0.125g lead citrate).