What kind of particle size should I use?
Always use the smallest particle size to fit your application. Conjugates based on smaller particles are more efficient than larger particle based conjugates. If visualization is difficult with smaller particles these can be enlarged with silver enhancement, which is a must for the conjugates from the Ultra Small series. The silver enhancement system AURION SE-EM provides for homogeneous and high efficiency enhancement.
Is it true that gold conjugates are more background prone than other conjugates?
No! This fairy tale comes from the fact that gold conjugates are based on particles and that visualization is also based on separate particles. Contrary to enzyme and fluorescent markers, gold conjugates are more like a digital system, either they are there and then you will see them, or they are not present. Enzyme and fluorescent markers are sooner to be considered as “analogue” markers, their visibility in detection increases with their local concentration or with the time the enzyme marker can produce a visible reaction product. An unbiased look at controls in fluorescence shows always a low level of light that is inherent to the presence of double bonds in biological compounds and on top of this comes the fluorescence from the labeled antibodies. Likewise will an unbiased look at control specimens incubated only with alkaline phosphatase or peroxidase labeled antibodies usually show a faint overall staining of the specimen. Such faint levels are easily accepted or even mentally filtered out. You cannot do this with gold conjugates since they are based on particles.
Should I use a secondary gold conjugate or Protein A (or G)?
That depends on what your goal is. Using secondary conjugates results in a higher labeling density. Therefore it is often said that secondary conjugates are more sensitive than Protein A conjugates. This is partly true. Protein A (or G) recognizes only one site on a primary antibody molecule. Binding will occur only when this site is available and not obscured by its environment. Secondary conjugates recognize more sites on primaries and therefor the chance that a primary antibody will be detected is greater. Essentially this is the increase in sensitivity.
In which case should I use a Single Fab or F(ab’)2 conjugate in stead of the complete immunoglobulin conjugate?
The size of a conjugate is co-responsible for its efficiency. The overall size is determined by the particle size and by the size of the proteins adsorbed onto the particle surface. That is why we introduced ultra small particles in the first place. Whenever a specimen is relatively dense or intensely cross-linked immuno reagents will be more hindered in their action. This is especially valid in pre-embedding immuno labeling (Newsletter #5). If you are already using an ultra small conjugate further improvement may result from using a single Fab or F(ab’)2 fragment of the specific secondary antibody instead of the intact IgG-molecule.
Is it advisable to use outdated conjugates?
As long as their reactivity is OK and there are not too many clusters formed this is no problem. Gold conjugates are very stable. There may be some release of protein from the particle surface with time, but generally this does not result in noticeably reduced reactivity. The reactivity of the conjugate is easily checked with a dot-spot test as described in Newsletter #4. Cluster formation may increase with time, depending on the type of conjugated protein and the particle size. The larger the particles the more clusters. These can be removed by centrifugation of the diluted conjugate before use.