Optimized
Labelling
Optimized Immuno Labelling using AURION BSA-c™ and AURION Blocking Solutions
How does the blocking step work?
When is a block step adequate?
Appropriate blocking of specimens is the first step towards optimized immuno labelling results. Multipoint interactions between specimen and immuno reagents can be prevented by a Blocking Step, using proper blocking agents that have both hydrophobic and hydrophilic properties. This can be achieved in many ways, e.g. with solutions containing BSA or Casein and is most effective at a pH-value close to or slightly higher than the iso-electric point of the blocking agent. These conditions leave the blocking agent with little net charge, thus favoring hydrophobic interaction. AURION Blocking Solutions contain a specially selected and preconditioned BSA for enhanced multipoint interactions. During the adsorption process the BSA molecules interact with a surface via many interaction points which can bind over a certain time span. For the molecules to be desorbed however, all those interactions would have to become released at the same time. The chance for this to happen is very small, so an adequate blocking step is (certainly in view of the time of incubations) virtually permanent. Once blocking of hydrophobic areas has occurred, the areas have been rendered hydrophilic. Figure 1 shows an interpretation of the events during the block step.
Figure 1: The blocking step
A: The blocking solution is applied.
B: Blocking compounds like globular BSA molecules adsorb onto the specimen surface. Initially the adsorbed globular molecules do not share many points of interaction with the specimen surface as indicated by the arrows: BSA molecules can still relatively easily become washed off the surface.
C: With time BSA molecules start to unwind so as to increase the number of interaction points: the blocking becomes less reversible.
D: At the completion of the blocking step, most BSA molecules will share many interaction points (small arrows) with the specimen, the result is a firm binding.
Important: There are still areas where specimen surface is exposed, as these are too small to be covered by large globular BSA molecules (large blue arrows with exclamation marks).
Is the blocking step sufficient for optimum signal-to-noise ratios?
Between the blocking components on the specimen surface there are areas left uncovered, as they are smaller than to allow a globular protein to fit in. Such areas may be large enough to accommodate markers or parts of antibodies, giving rise to background. Whether this will actually happen also depends on the characteristics of the specimen exposed in these areas: if they are positively charged or hydrophobic they will tend to bind antibodies and labels, resulting in serious background problems. Therefore, after the block step an incubation solution has to be used that should be serving two purposes:
- creating an environment promoting antibody-antigen binding and
- providing a means to control the left-open spaces on the specimen surface.
This is easily achieved with an incubation solution containing BSA-c™.
