The conjugates in the BBI range are the most popular secondary labelled antibodies. However, we do have a much larger range of antibodies available for conjugation to any of the standard gold or silver particle sizes. The cost of these conjugates is higher than our standard products as they are made to order, but lower than a custom conjugate using a customer’s antibody as we are familiar with them and can process them quickly because of the short development time. We have several hundred antibodies available raised in different species against many immunoglobulins and absorptions. Please contact our technical department for prices and availability on tech@bbigold.com.

BBI have a range of colloids and protocol for those who would like to conjugate their own antibodies. Initially this may seem the most cost effective way of producing a conjugate, but if you have little experience of producing a conjugate, you may find the cost in time and material developing a stable, sensitive conjugate, mounting up. BBI offer an alternative to this – a custom conjugation service. Through many years of experience, BBI can take your antibody and produce a sensitive conjugate within a short time frame. We can also give advice on what is likely to work and what is unlikely. Please contact out technical department for advice and cost of this service on tech@bbigold.com.

The aim of an antibody incubation with a specimen, whether it is a section, cells in suspension, a tissue slice, a blotted membrane, or a rapid diagnostic test assay, is to achieve the most intense specific signal and the least non specific background possible.

Gold Conjugates may be used directly or indirectly to label antigens as already indicated. In the direct method the gold is conjugated to primary antibody, whether monoclonal or polyclonal. This allows a single incubation to be performed and provides the simplest detection system. In the indirect method a primary unlabelled antibody is applied to the spectrum to locate the antigen. This is followed by the gold secondary antibody that detects the primary antibody. This gold labelled secondary antibody is almost always affinity purified polyclonal.

An extension of the indirect method may be used where there is no appropriate gold labelled secondary to match the primary antibody. For example, if the unlabelled primary is from Pig and there is no matching gold labelled anti-Pig then an unlabelled anti-Pig (sat Rabbit anti-Pig) may be used as a secondary step followed by a gold labelled third antibody (say Goat anti-Rabbit) that will detect the second antibody.

The indirect method is the most common for studying cells and tissues and avoids the need to label every primary antibody. It provides a universal method for detecting any primary antibody from the same primary species, i.e. all Rabbit antibodies may be detected by Goat anti-Rabbit gold conjugate, etc. The indirect method is longer than the direct method because of the need for two (or three) separate incubations but is the more widely used.

Antibody gold conjugates are absorbed against certain species of serum proteins as detailed in the Product Section. This absorption reduces certain reaction to those species to a minimum.

In some applications background labelling may be a problem due to the attraction of the Fc region of the antibody gold conjugate to tissue components (called Fe receptors). Normally this is blocked by the simple application of normal serum prior to the first antibody. If the problem persists, however, then gold labelled F(ab’) fragments of antibodies may be used. These conjugates are listed in each section.

Protein A (PAG), Protein G (PGG) and Protein A/G (AG) are all separate proteins and will mimic a secondary antibody by binding the Fc part of the primary antibody. In some situations they may be used in place of a secondary antibody. Unlike secondary antibodies, however, it is believed that there is only a single binding site for these proteins on the primary IgG. They so not bind with great affinity to IgM or IgA molecules. The advantage of these proteins is that they provide a universal second step for a wide range of primary antibody species. They each bind with varying affinities to IgG molecules of different species as shown in the table. However, if possible we would always recommend the selection of species specific secondary antibody in preference to Protein A, G or A/G.

For the indirect detection of a biotinylated primary antibody or nucleic acid probe there is a choice of Streptavidin or Goat anti-Biotin gold conjugates. Historically, Streptavidin has been the most frequently used detector for biotin because of the very high affinity constant between them. Most recently, however, Goat anti-Biotin has been shown to be a rather more sensitive detector of biotin compared to Streptavidin when conjugated to gold particles. This is because of the relatively large molecule size of the antibiotin molecule (160,000 Daltons) compared to Streptavidin (40,000 Daltons) and the distance between the binding of the gold on the Fc, from binding region of the antibody F(ab’). This is especially so when using larger gold particles but becomes insignificant for 5nm and 1nm gold conjugates.

On occasions where background from non-specific attraction of goat antibodies may cause a problem, Streptavidin may provide a cleaner result. We recommend beginning with a Goat anti-Biotin gold conjugate for the detection of biotin.

Cationic gold allows highly sensitive and discrete microscopical studies of anionic (i.e. negative) sites in cells and tissues. The gold conjugate is made by careful coupling of poly-L-lysine, a highly positive amino acid chain. A simple of step incubation of sections with the diluted Cationic Gold conjugates reveals subcellular sites having net negativity charge. The charge distribution can be seen at a range of magnifications by using cationic Gold of different particle sizes.

(Reference:
1. Bush MS and Allt G (1990) “Blood nerve barrier distribution of anionic sites on the endothelial plasma membrane and basal lamina” Brian Res. 535. 181188.)

A wide range of particle sizes can be conjugated to proteins and macromolecules. In principle, smaller gold particles produce a higher labelling intensity on the specimen. This is because of the reduced steric hindrance to antigen detection. Typically an antibody of 160,000 Daltons molecular weight will have a linear dimension of 8nm. Thus a 1nm gold particle, attached to the fc region, will hardly impede the antibody activity. A 20nm particle, however, while being more visible, will produce a greater steric hindrance by its proximity to the antigen binding region of the antibody. In addition, the increased charge repulsion between larger particles reduces the number of labelled antibodies gaining access to the target antigen.

Different particle sizes are appropriate for different types of application as described below.

a) Light Microscopy

Gold particles cannot easily be seen in the light microscope by bright field viewing. They must, therefore, be silver enhanced for greater visibility. Small gold particles will give the greatest number of gold labelling on the specimen and each particle can then be subsequently silver enhanced for maximum visibility of signal. A choice can be made between 1nm and 5nm gold conjugates. The 5nm conjugates are used for most standard purposes and are recommended for the initial studies. Foe even greater labelling intensity the 1nm gold conjugates are particularly useful when penetration into cells and tissues (e.g. pre-embedding labelling) is required.

b) Transmission Electron Microscopy

For electron microscopy, any particle size may be employed. For low magnification work, larger particles (e.g. 15 to 30nm) are more easily seen. For high magnification studies, the smaller particle (5 to 10nm) are preferred, Again, smaller particles give higher labelling intensity and may be subsequently silver enhanced on the section to produce larger particles with this high labelling intensity. For ultra high sensitivity, the 1nm gold particles can also be chosen but are usually visualised only after silver enhancing on the section. For those just beginning immunogold labelling in the EM or performing studies over a range of magnifications, 10nm gold conjugates are recommended.

c) Scanning Electron Microscopy

The resolution of the screening electron microscope indicates that larger particles (e.g. >20nm) should be used for detection by back scattering electron signals. However, as mentioned above, a greater intensity of labelling is achieved with a smaller gold particle, which can be subsequently silver enhanced with LM/EM Silver Enhancing Kit. We would therefore recommend that a choice is made for SEM studies between 20 or 30nm gold conjugates for direct (un-enhanced) viewing, or 5nm conjugates which can be simply silver enhanced within minutes for observation at low magnifications. With the latter approach each gold particle grows spherically to approximately 30nm in 35minutes. The reaction is simply stopped by washing in water.

d) Blotting

For blotting applications where the gold conjugates are to be applied to proteins immobilised on nitrocellulose membranes, the choice is between 1nm & 20nm BL grade gold conjugates. Even 5nm conjugates may also be used very effectively (LM grade) together with subsequent silver enhancing. For ultra high sensitivity, 1nm conjugates are preferred in combination with silver enhancing. However, the 20nm gold conjugates allow blotted proteins to be identified on the immobilising membrane without silver enhancing. The 1nm and 5nm (LN grade) gold conjugates, because of their much reduces steric hindrance and greater particle density in solution, may be diluted much further to provide the same final intensity compared with the 20nmc conjugates. The 1nm and 5nm (LM) gold particles mist be enhanced with the Blotting Silver Enhancing Kit.

For further information on any of the above issues please contact tech@bbigold.com

Conjugates stored at 4°C will be stable for one year from purchase. The life can be extended by aliquoting and storing at -20°C (repeated freeze / thawing should be avoided as this will reduce conjugate activity).

If a conjugate has been stored for a long time without use, it is advisable to check it is still sensitive. The simplest method is to dot blot 1µl aliquots of the antigen in a series onto nitrocellulose. 5 and 10nm particles should detect 10ng/µl: and 15 and 20 nm about 5ng/µl. 1nm needs to be silver enhanced and should detect 10pg/µl.

Yes. For more information, please contact our technical team on tech@bbigold.com.

5nm and 10nm will give a clear difference, but 5nm and 10nm is unequivocal. When choosing the particle sizes, it should be noted the small particle size will give a higher labelling.

Smaller particle sizes will give the best labelling, but at low magnifications in the EM, larger particles will have to be used. For more information on Gold Conjugates please look at “Gold Conjugate Technical Information” in the literature section.

For more information on Gold Conjugates please look at “Gold Conjugate Technical Information” in the literature section.

Buffer choice, pH, dilution and blocking reagents all control background. Contact tech@bbigold.com for more information on controlling background.

Controls serve two purposes. They show our experiment has worked correctly and can help identify the cause of a problem when it doesn’t. Contact tech@bbigold.com if you require suggestions on which controls to sue for your application. For more information on Gold Conjugates please look at “Gold Conjugate Technical Information” in the literature section.

Nickel or gold grids are best, copper grids are not advisable. (Note: if using grids, don’t try silver enhancing).

Epi-polarised light is used for the sensitive light microscope observation of metal particles. Larger silver enhanced particles can be individually observed.

Protein binds to gold colloid for three main reasons: charge (lysine), hydrophobic attraction (tripophan), and sulphur bonding (cystine and methinine). Generally the attraction isn’t reversible, but long term storage at high pH or in a buffer containing surfactant may cause some protein to become unbound.

No. In fact it is the opposite. Proteins that have been absorbed against antibodies from other species shouldn’t work against them. This is to prevent cross labelling when doing double labelling.

F(ab’)2 fragments can be used when background is a problem. 1nm F(ab’)2 fragment conjugates should also give better cell penetration than whole molecule 1nm conjugates.

These can be used when the researcher is using antibodies from different hosts when only a single conjugate needs to be purchased. The disadvantage of using these proteins is that labelling efficiency is lower than with antibody specific conjugates.

No. Protein AG is a single marker that labels a wider range of host animals than either Protein A or Protein G. For more information please contact our technical team on tech@bbigold.com.

Most users report anti-Biotin conjugates give superior labelling to Streptavidin and we would recommend anyone trying a biotinylated system for the first time, to use anti-Biotin. We have found higher labelling with Streptavidin at pH5, but we would advise caution at this pH with regards to non-specific labelling.

It is not possible to remove all of the Glycerol but the amount of glycerol can be reduced so that it is no longer a problem. The conjugate can be dialysed against TBS or PBS without glycerol with two changes of buffer. This will reduce the amount of glycerol with two charges of buffer. This will reduce the amount of glycerol present in the buffer quite considerably. Centrifuging the conjugate and re-suspending in a TBS or PBS buffer with 1% BSA is quicker, but may introduce clustering. Test a small, diluted volume first.

Labelling intensity is higher with small particles. Small particles also sliver enhance better than large particles. 40nm and above practically don’t enhance.

The choice is between 1nm and 20nm. Labelling with 1nm but has to be enhanced to be visible. 20nm can be seen without enhancement. Other particle seizes can be used, and EM 40nm will give good results without enhancement.

The colloid used to make the EM products is of the highest quality and has a tight size and %CV specification. Therefore the image in the EM is of a uniform particle size. The LM conjugates may have some small particle groups and the image in the EM will not look so good. There may also be some overlap with the 10nm particle size. It is possible to interchange the conjugates. LM particles will work in the EM and EM in the LM, but when using LM in an EM application, the user should be aware the image won’t be as good.

Colloid used for Blotting Grade is not of a high enough quality for EM work. The particle size range is quite wide.

We did have a range, but we found it difficult to reproduce the high quality batches that customers expect of BBI conjugates and we no longer feature them in our catalogue. We will make them as a custom conjugation, but for most applications we recommend using biotinylated lectins and use Goat anti-biotin as the marker. Biotinylated lectins are available from Vector Laboratories Inc.

Goat anti-Biotin, Streptavidin and Sheep anti-Digoxigenin are used for In-Situ Hybridisation. For further information on any of the above issues please contact tech@bbigold.com

Gold colloid will keep at 4°C for 6 months in an unopened bottle. After opening we recommend that the colloid is conjugated within one month

Store the colloid at 4°C. Use at room temperature. Colloids opened in a laminar flow hood will remain stable longer than colloids opened on a bench. Colloids cannot be frozen. Even partial freezing of the colloid will cause the colloid to precipitate, leaving a clear solution. Colloids damaged by freezing cannot be used and will not be replaced by BBI under guarantee.

The principle of making a conjugate is simple; forces attract the protein and the colloid together. A protocol is included with the colloid on how to make a conjugate, but even so, most inexperienced operators find conjugate manufacture difficult. Unless you have some experience in this you may find it quicker and more economical to use BBI’s Custom Conjugation Service.

No. BBI’s gold and silver conjugates are not suitable for injection or by mouth. BBI do not accept responsibility for misuse of any of their products.

BBI’s colloid was developed for use as a label for conjugation to small molecules. It may be suitable for other applications; this is for the user to decide. BBI does not offer support to applications outside the intended use of the colloid (conjugation).

BBI’s colloid manufacture methods have been developed by our scientists over many year and as such that are proprietary to the company. The methods we use ensure very high quality and repeatability over the range to allow us to make top quality conjugates.

The starting point is a good colloid, but control overall of the parameter during conjugation will give a stable conjugate that performs well.

The largest particle size we manufacture in gold is 250nm and in silver 80nm. We may make larger particles to order. Please contact our technical department for details on tech@bbigold.com

The colloid has an overall negative charge, which keeps it in suspension. This charge is also part of the reason why the colloid binds protein. The net positive charge of a protein (lysine), coupled with hydrophobic binding (triptophan) and sulphur bonding (cystine and methinine) causes the attachment between the colloid and protein.

Not at this time but it is something we are considering for the future.

The colloid is in water (as made), with only residual chemicals left from manufacture. It is not stabilised, the charge on the particles keep them in suspension. The weight of larger particles is too much for the charge and eventually these sink out of solution. Colloids that have settled in this way can be resuspended by inversion.

No. Frozen colloids precipitate and become a clear solution. Ideally colloids should be stored in the door of a fridge or away from the chillers in a cold room to prevent accidental freezing. Colloids that have been frozen will not be replaced as part of the BBI guarantee.

There is no easy way to do this. On a small scale, boiling will reduce the volume of fluid and is probable the only way to achieve this, but there are limits, as chemicals from manufacture will be concentrated too. Centrifugation is only possible after conjugation, as an uncojugated colloid will form precipitate that cannot be resuspended. Some colloids can be made at a higher concentration.

For further information on any of the above issues please contact tech@bbigold.com