In immunofluorescence technique, one of the steps in the procedure is the blocking step. We add blocking solution (universal protein block) before we introduce our antibodies, and incubate 30 mins. However, how do we know that blocking solution does not block the specific binding site on the protein of interest that we want to observe? Since we do not use blocking solution specific to each different antibody, and use only one blocking solution in all our experiments, how do we predict that blocking reagent does not block the specific binding site before we add any primary antibodies?
Thank you!
As not an expert in the field of immunofluorescence and not having particular experience;
I may opine on the subject based on immunoblotting. Typically used blocking agents such as non-fat dry milk, BSA, and detergent-based commercially available reagents may have this drawback. Even if the primary purpose is blocking the unoccupied and reactive sites, they can also interact with the POI. However, if the conjugate (POI and blocking protein) is not a kind interaction/binding partner (considered like co-IP), immunoaffinity would present a stronger and selective interaction and possibly displace the weakly bound (non-specifically bound) blocking protein.
For instance;
Albumin in serum interacts with the isolation nano-beads (magnetic beads at proteomic investigation/ pulldown assays) and forms a
Protein corona temporarily on beads due to their bulky abundance. In seconds proteins in serum have more avidity to the bead ligands are replaced with albumin, thus both albumin depletion and target protein isolation can be achieved (look for seer proteograph and proteominer of bio rad)...
Immunoaffinity chromatography also works this way. Elution is done either by flowing a more interactive affinity ligand to neutralize binding or by increasing the concentration of the less interactive ligand.
In summary, I may assume this potential issue is overcome in many cases by immunoaffinity interaction-based replacement provided by primary antibody...
Blocking solutions use proteins like bovine serum albumin (BSA) or serum from the host species of the secondary antibody. These are "passive" proteins that act by coating the sample, not by specific binding.
The blocking solution does not contain antibodies that would bind to the specific target antigens. The primary antibodies are introduced after blocking precisely so they can access the specific binding sites. Also, the primary antibodies are used at a relatively high concentration compared to what would be needed to saturate all the binding sites. So even if some sites are blocked, there are still plenty of unblocked sites for the primaries to bind.
The blocking step is timed to saturate the sample, but not long enough to displace specifically bound antigens. 30 mins is typically enough time to block non-specific sites without interfering with specific binding.
However, If blocking does inadvertently block some specific sites, then the signal may be reduced, but it does not necessarily abolish binding completely.
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication