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Blocking Solutions Application Instructions Detailed information is provided on the package inserts.
Electron Microscopy On-Grid Labeling
Placing grids on top of drops of Blocking Solution arrayed on a sheet of Parafilm™ after they have been treated for aldehyde inactivation.
Floating specimens in Blocking Solution, preferably on a rocking table. Light Microscopy
On-slide or -coverslip Labeling
Applying a few drops of Blocking Solution to cover the specimen. Bio Assays
Incubating specimens (e.g. immunoblot strips) in Blocking Solution on a rocking table in screw-cap sealed disposable tubes.
For additional information please see the Aurion Immunogold Newsletter and Protocol sections.
The signal-to-noise ratio determines the quality of any detection experiment. This ratio is in principle determined by the characteristics of specimen and detection reagents. With adequate blocking and incubation media, background reactions are minimized whereas specific reactions are not hampered.
Aurion has developed Blocking Solutions which effectively block ‘sticky’ surfaces by multi-point hydrophobic and charge-based interactions. The Blocking Solutions are specifically designed to meet the characteristics of each type of secondary conjugate.
The AURION Blocking Solutions are used to prevent immunoreagents from binding non-specifically to specimens with “sticky” surface properties.
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
N-Cadherin detection in heart muscle cells
Immunofluorescence using Alexa 568 labeled Fab goat antimouse. For reasons of comparability areas of specific labeling are pictured with similar density (arrowheads).
Courtesy of Lauren Hruby and John Harris; Dept. Physiology, University of Otago, Dunedin, New Zealand Left hand panel:
Right hand panel:
Background using a commonly used protocol obscures sites of specific labeling.
N-Cadherin immunolabelled areas obtained using Aurion Blocking Solution and BSA-c™ stand out with much clearer definition.
To reduce aspecific 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. These steps should be balanced for optimum results.
Product D escription
AURION Blocking Solutions are prepared using specially selected compounds. All ruminant proteins are obtained from healthy livestock.
AURION Blocking solutions contain Bovine Serum Albumin and Cold Water Fish Skin Gelatine in phosphate buffered saline with sodium azide as preservative. Normal serum may have been added as indicated on the label. The blocking capacity of each lot is determined using a dot- spot test system as described by Moeremans et al., J. Immunol. Methods 74, (1984), 353.
Each package contains 30 ml of solution. It accommodates 300 specimens for light microscopy at 100 µl/specimen (~ 3 drops), or 1000 EM grids at 30 µl/specimen (~ 1 drop).
Specificity Blocking Solutions are available in the following specificities:
serum-free: for use with Protein A and Protein G Gold conjugates. with Normal Goat serum: for use with reagents based on secondary antibodies raised in Goat.
with Normal Rabbit serum: for use with reagents based on secondary antibodies raised in Rabbit.
with Normal Sheep serum: for use with reagents based on secondary antibodies raised in Sheep.
with Normal Donkey serum: for use with reagents based on secondary antibodies raised in Donkey.
AURION Blocking Solutions have 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.