In the blocking step, hydrophobic moieties causing "stickiness" in the specimen surface are rendered hydrophilic to minimize background. Nevertheless, the more dynamic charge-based interaction between the specimen surface and immunoreagents also needs to be controlled in order to eliminate background.
Aurion has developed BSA-c™, a unique incubation buffer additive with an unparalleled ability to effectively prevent charge based background. BSA-c™ is prepared by acetylation of bovine serum albumin (BSA). Polycationic sites in the specimen interact readily with negatively charged acetylated BSA molecules. This significantly reduces the risk that such sites might bind negatively-charged immunoreagents and immunogold conjugates and thus reduces the risk of background.
Procedures to eliminate background comprise three main steps:
- To suppress residual aldehyde activity
- To saturate multipoint hydrophobic moieties and positive charges with high molecular weight compounds such as those present in the AURION Blocking Solutions
- To reduce non-specific binding of immunoreagents caused by hydrophilic interaction with competing molecules in the incubation and washing solution.
AURION BSA-c™ is a particularly effective reagent for this purpose.
AURION BSA-c™ is a buffer additive that helps prevent immunodetection reagents (i.e. primary antibodies and secondary reagents) from binding nonspecifically to charged moieties within the specimen. Thus, it suppresses background competitively with little or no effect on the specific reaction. Its successful application is not limited to immunogold detections but it is equally efficient in fluorescent and enzyme-based detection systems. AURION BSA-c™ concentrations as low as 0.01-0.1% inhibit binding of gold conjugate to polycationic poly-l-lysine coated grids almost completely (>99%).
The surface properties of the specimen can be simplified by division into four compartments:
- negatively charged (polyanions, proteins, especially after aldehyde fixation, lipids);
+ positively charged (histone proteins, polycations) and H hydrophobic (lipids, fat droplets, resins). After an appropriate blocking step these areas are covered with blocking compounds.
In low ionic strength media negatively charged antibodies and gold conjugates are repulsed by negatively charged specimen areas which frequently may contain the antigens to be detected. Background does not likely occur in such areas. The positively charged areas attract antibodies and gold conjugates potentially leading to background. In a moderate ionic strength incubation solution, repulsion and attraction are diminished due to the presence of ions. The negatively charged BSA-c™ competes with the negatively charged antibodies and gold reagents for non-specific binding to the positively charged specimen compounds, thus reducing background to the greatest possible extent without interfering with antigen detection.
Light microscopy evaluation of tubulin labeling with Ultra Small Immunogold Reagents and silver enhancement. Upper panel: bright field mode Lower panel: epi-polarization mode
AURION BSA-c™ concentrated solution contains acetylated bovine serum albumin as the functional constituent. By acetylation of amines on basic amino acids these groups are no longer as easily protonated and the isoelectric point of such molecules is lowered and hydrophobicity is increased. BSA-c™ is a 10% solution of acetylated BSA at slightly alkaline pH with Kathon CG as preservative. The bovine serum albumin that Aurion uses to prepare BSA-c™ is obtained from healthy livestock.
The charge dependent background inhibition capacity of the BSA-c™ in each lot is determined using a dot-spot test system with polycationic compounds.
Two package volumes are available which yield 1.5 - 3 and 5 -10 liters of incubation buffer respectively
AURION BSA-c™ concentrate has a guaranteed shelf life of 18 months from the date of quality control analysis. The products should be stored at 4-8°C. Freezing is not recommended.
Incubation Solution Additive AURION BSA-c™
Controlling Background in Immunogold Labeling