Streptavidin coated surfaces offer a powerful and universal instrument for binding any biotinylated molecule (Proteins-Peptides-Polysaccharides-Oligonucleotides-DNA fragments-etc.)
Streptavidin is a tetrameric protein (M.W. 60 kDa) with very high affinity for biotin (Ka=10 –15M); the bond is the strongest known non-covalent biological interaction.
Biotin is a small molecule which can be conjugated to many proteins without loosing or altering their activity, each protein can bind many biotin molecules.
Since each subunit of streptavidin binds one molecule of biotin, the resulting effect is a great increase of the sensitivity of the assay.
The streptavidin-biotin bonding main features:
- stability
- specificity
- affinity
make it useful for special applications of molecules which do not offer reliable bonding by passive adsorption or adsorb in an unfavourable orientation.
Available configurations
Uniformity
The coated products show a CV% less than 5 using biotin-HRP as detector and TMB as substrate.
Storage and Stability
The Streptavidin coated products, if unopened, are stable at 2-8°C until the expiration date printed on the label. If opened, store in closed pouch with desiccant and use within the expiration date.
Product specifications for microplates
Coating
Streptavidin is coated using 200 µl/well. The strips are post-coated (blocked) for low non specific binding and long-term stability.
Functional features of streptavidin coated plates.
The following parameters were analysed:
- binding capacity towards biotin
- specificity towards biotin
- binding capacity towards biotinylated IgG
- stability tests:
- endurance under strong chemical contacts
- shelf life at 37°C
- temperature stress (transport simulation)
- long storage
- binding capacity towards biotin
Streptavidin coated wells (and BSA saturated control wells ) were incubated with a calibrated biotin solution.
Subsequently, aliquots of this solution, concomitantly with biotin standards, were mixed with biotinylated peroxidase and transferred into new empty streptavidin coated wells. From the amount of enzyme bound to the solid phase, the biotin content of the samples was calculated.
This value was compared with the amount of biotin originally added; from the difference (corrected for-non specific binding of biotin to the control wells), the capacity of the wells for biotin was derived.
12 pmol/ well (200 μl volume)
Specificity towards biotin
Streptavidin coated wells were incubated with solutions of biotinylated peroxidase and unbiotinylated peroxidase (blanks) as a catcher in a ELISA format using TMB as substrate.
Binding capacity towards biotinylated HIgG
Streptavidin coated wells were incubated with biotinylated HIgG in an ELISA format using HIgG-HRP as a detector and TMB as substrate.
100 ng/ well (100 μl volume)
Stability tests:
Endurance under strong chemical contacts
Endurance under strong chemical contacts was determined following method 1 (Biotinylated IgG from 500 to 0 ng/ml) where the first washing step was substituted by the following incubations:
| chemical compound | conditions |
|---|---|
| SDS 0.1% 0.6M NaCl | 56°C 2h |
| 30% Formamide in 0.6M NaCl | 37°C 2h |
| 0.1% Tween® 20 in 0.1M PBS | 37°C 2h |
| 0.05M NaOH | RT 20' |
| 0.2M NaOH | RT 20' |
| standard - 0.1% Tween® 20 in 0.1M PBS | 4 x |
| Urea 8M | 60'-30'-15'-5' |
Shelf life at 37°C
Streptavidin coated wells maintained for 15 days at 37°C in comparison with standard stored at 4°C,
Long storage
Streptavidin coated wells were maintained for 30 months in a warehouse without air conditioning (temperature range from 10°C to 40 °C ) in comparison with samples stored at 4°C (standard condition ).
The results show the exceptional stability of the coating.
Temperature stress (transport simulation)
Stability was checked under different temperatures as those which may occur during transport.
| plate n. | conditions | time |
|---|---|---|
| 1 | 4° C | 10 days |
| 2 | 22°-23° C | 10 days |
| 3 | 37° C 22°-23° C - 20° C 37° C 22°-23° C | 3 days 12 hours 10 hours 3 days 3 days |
Streptavidin Coated Available Microplates
Product specifications for PCR 8 strip tubes and plates
Coating
Streptavidin is coated using 100 µl/tube. The PCR 8 strip tubes and plates are post-coated (blocked) for low non specific binding and long-term stability.
Binding capacity towards biotin
Streptavidin coated PCR tubes strips were incubated with biotin solutions containing biotinylated peroxidase for 30’ R.T. After the washing step, the PCR tubes strips were incubated with TMB and blocked with sulphuric acid 1N. The O.D. values were read at 450 nm
~ 5-6 pMol d-biotin/tube
Streptavidin Coated Available PCR Tubes
Product specifications for 12×75 mm tubes
Coating
Streptavidin is coated using 500 µl/tube. The tubes 12×75 are post-coated (blocked) for low non specific binding and long-term stability.
Binding capacity towards biotin
Streptavidin coated tubes 12×75 were incubated with biotin solutions (from 0 to 195.2 ng/ml) containing 4 ng/ml of biotinylated peroxidase for 30’ R.T. After the washing step, the streptavidin tubes 12×75 were incubated with TMB and blocked with sulphuric acid 1N. The O.D. values were read at 450 nm.
~ 28 - 30 pMol d-biotin/tube
