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:
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 litres of incubation buffer respectively
Detailed information is provided in the package inserts.
Recommended Incubation Solution:
(10 mM Phosphate buffer, 150 mM NaCl)
0.1-0.2 % AURION BSA-c™
15 mM NaN3
After addition of the BSA-c™ concentrate the pH should be checked and adjusted to 7.4 if necessary.
This Incubation Solution is used throughout the whole set of reactions and washing steps (i.e. from immediately after the blocking step, through the primary antibody incubations, in-between washing steps, secondary incubation and the final washing steps).
Note on background prevention: A special AURION Newsletter on background is available upon request.
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.