Many compounds have been added empirically to immunolabeling solutions for the purpose of minimizing background staining. Based on years of experience and controlled testing, Aurion has selected a group of compounds that are proven to be the most effective in their background reduction action. Researchers can obtain these items separately from Aurion and prepare blocking and incubation media to suit the needs of their own protocols.
Introduction
The ready-to-use Blocking Solutions and the incubation media additive BSA-c™ are tuned for optimum background prevention and signal-to-noise ratio. In-depth information can be found in the respective product data sheets.
AURION also offers a number of components that allow researchers to formulate blocking, incubation and wash solutions according to the needs of their own experiments.
Product Description
Aurion’s Bovine Serum Albumin is obtained from healthy livestock. BSA should be dissolved in an appropriate buffer, such as phosphate buffered saline, taking care not to denature the protein by foaming. The addition of BSA may cause a drop in pH of the final solution and correction may be required. As a preservative the use of NaN3 or Kathon CG is recommended.
The use of Cold Water Fish Skin Gelatin to prevent background reactions has been recommended by e.g. Behnke et al. (J. Cell Biol. 41, [1986], 386). The product is supplied as a liquid concentrate (40%).
Tween-20™ is a non-ionic detergent with a molecular weight of about 600 and a critical micelle concentration (CMC) of 0.06-0.07% in water at room temperature. Its working mechanism may in part be based on its action as a detergent, binding to the hydrophobic moieties of water insoluble compounds, rendering them hydrophilic. In addition, immuno-compounds may become incorporated into micelles when the Tween-20™ concentration is higher than the CMC, for instance at 0.1 % in PBS at pH 7.4.
Normal sera are used to counteract the non-specific interaction between the sample and immunoglobulins. They can be added to the blocking solution and the incubation media.
As a rule the normal serum species should be the same as the secondary antibody species (e.g. use normal goat serum with goat-anti-rabbit conjugates).
Note: normal sera should not be used in combination with Protein A and Protein G gold reagents.
Application instructions
The following recipes have been tried succesfully by researchers in the field and by Aurion:
Blocking Solution for protein A or G reagents
Phosphate buffered saline
(10mM Phosphate buffer, 150 mM NaCl)
5 % BSA
0.1% CWFS Gelatin
15 mM NaN3
pH 7.4
Blocking Solution for secondary antibody reagents
Phosphate buffered saline
(10mM Phosphate buffer, 150 mM NaCl)
5 % BSA
0.1% CWFS Gelatin
5-10 %normal serum
(same species as in the secondary antibody reagent)
15 mM NaN3
pH 7.4
Incubation solution for Conventional Immunogold Reagents
Phosphate buffered saline
(10mM Phosphate buffer, 150 mM NaCl)
0.1-0.2 % BSA
0.1% CWFS Gelatin
1-5 %normal serum
(same species as in the secondary antibody reagent)
15 mM NaN3
pH 7.4
Incubation solution for Ultra Small Immunogold Reagents
Phosphate buffered saline
(10mM Phosphate buffer, 150 mM NaCl)
0.8 % BSA
0.1% CWFS Gelatin
1-5 %normal serum
(same species as in the secondary antibody reagent)
15 mM NaN3
pH 7.4
Note on background prevention:
A special AURION NEWSLETTER on the subject of background is available upon request.
Storage
The products should be stored at 4-8°C.
Freezing is not recommended.