Dynabeads® M-280 Streptavidin are ideal for numerous applications, including purification of proteins and nucleic acids, protein interaction studies, immunoprecipitation, immunoassays, phage display, biopanning, drug screening, and cell isolation.
Add Dynabeads® to a sample containing biotinylated molecules e.g., peptides, proteins, antibodies, sugars, lectins, oligonucleotides, DNA/RNA. During a short incubation, the biotinylated molecule will bind to the beads. Separate the molecule-bead complex with a Dynal® magnet. Capture, washing and detection can be optimized for manual or automated use. With indirect capture, mix the biotinylated molecule with the sample to capture the molecule-target complex before adding Dynabeads®. Indirect target capture is an advantage when molecule-target kinetics are slow, affinity is weak, molecule concentration is low or molecule-target binding requires optimal molecule orientation and true liquid-phase kinetics.
Description of materials
Dynabeads® Streptavidin are uniform, superparamagnetic beads of 2.8 μm in diameter with a streptavidin monolayer covalently coupled to the surface. This layer ensures negligible streptavidin leakage while the lack of excess adsorbed streptavidin ensures batch consistency and reproducibility of results.
Dynabeads® M-280 Streptavidin are supplied as a suspension containing 10 mg (6–7 x 108)
Dynabeads® per mL, dissolved in phosphate buffered saline (PBS) pH 7.4, containing 0.1%
BSA and 0.02% NaN3 as preservatives. Available in three volumes:
Additional materials required
Magnet for manual or automated protocols. See www.lifetechnologies.com/magnets-selection for recommendations.
Table 1. Recommended buffers and solutions.
|For coupling of nucleic acids||For Dynabeads® treatment before RNA manipulations||For coupling of protein or other molecules|
|Binding and washing (B&W) Buffer (2x):||Solution A:|
|PBS buffer pH 7.4 These buffers can also be used for your application if needed:|
Additional material needed
All reagents used should be analytical grade. Isotonic (PBS) pH 7.2–7.6 We recommend the following PBS (pH 7.4):
PBS/BSA: Add 0.1% BSA fraction V (final conc.) to PBS. 0.02% NaN3 may be added as a preservative for storage, if needed.
Both the size of the molecule to be immobilized and the biotinylation procedure will affect the binding capacity. Large as well as small biotinylated molecules can be immobilized. The capacity for biotinylated molecules depends on steric availability and charge interaction between bead and molecule and between molecules. There are two or three biotin binding sites available for each streptavidin molecule on the surface of the bead after immobilization.
Recommended Washing Buffers
Calculate the amount of beads required based on their binding capacity (see Table 2), and transfer the beads to a new tube.
1. Resuspend the Dynabeads® in the vial (i.e. vortex for >30 sec, or tilt and rotate for
2. Transfer the desired volume of Dynabeads® to a tube.
3. Add an equal volume of Washing buffer, or at least 1 mL, and mix (vortex for
5 sec, or keep on a roller for at least 5 min).
4. Place the tube on a magnet for 1 min and discard the supernatant.
5. Remove the tube from the magnet and resuspend the washed Dynabeads® in the
same volume of washing buffer as the initial volume of Dynabeads® taken from
the vial (step 2).
Table 2 - Typical binding capacities for one mg of Dynabeads®.
|Free Biotin [pmol]||650 – 900|
|Biotinylated peptides [pmol]||~ 200|
|Biotinylated antibody [μg]||up to 10|
|ds DNA [μg] *)||~ 10|
|ss oligonucleotides [pmol] *)||~ 200|
* Oligonucleotides and DNA fragments
For oligonucleotides, capacity is inversely related to molecule size (number of bases). Reduced binding capacity for large DNA fragments may be due to steric hindrance.
Dynabeads® for RNA Manipulation
As Dynabeads® Streptavidin are not supplied in RNase-free solutions, perform the following steps after washing for RNA applications:
1. Wash the beads twice in Solution A for 2 min. Use the same volume of Solution A
as the initial volume of Dynabeads® taken from the vial or larger.
2. Wash the beads once in Solution B. Use the same volume of beads as in step 5.
3. Resuspend the beads in Solution B.
The beads are now ready to be coated with the biotinylated molecule of your choice.
Wash the Dynabeads® according to “Wash Dynabeads®” section before use.
1. Add the biotinylated molecule to the washed Dynabeads®.
2. Incubate for 15–30 min at room temperature with gentle rotation of the tube.
3. Place the tube in a magnet for 2–3 min and discard the supernatant.
4. Wash the coated beads 3–4 times in washing buffer.
5. Resuspend to desired concentration in a suitable buffer for your downstream use.
Here are some examples of immobilization protocols for specific applications.
Immobilize Nucleic Acids
1. Resuspend beads in 2X B&W Buffer to a final concentration of 5 μg/μL (twice original volume).
2. To immobilize, add an equal volume of the biotinylated DNA/RNA in distilled water to dilute the NaCl concentration in the 2 B&W Buffer from 2 M to 1 M for optimal binding.
3. Incubate for 15 min at room temperature using gentle rotation. Incubation time depends on the nucleic acid length: short oligonucleotides (<30 bases) require max. 10 min. DNA fragments up to 1 kb require 15 min.
4. Separate the biotinylated DNA/RNA coated beads with a magnet for 2–3 min.
5. Wash 2–3 times with a 1X B&W Buffer.
6. Resuspend to the desired concentration. Binding is now complete. Resuspend the beads with the immobilized DNA/RNA fragment in a buffer with low salt concentration, suitable for downstream applications.
1. Incubate the beads and biotinylated antibodies in PBS for 30 min at room temperature using gentle rotation.
2. Separate the antibody-coated beads with a magnet for 2–3 min.
3. Wash the coated beads 4–5 times in PBS containing 0.1% BSA.
4. Resuspend to the desired concentration for your application.
Release Immobilized Biotinylated Molecules
The biotin-streptavidin bond is broken by harsh conditions. 5 min incubation at 65°C or 2 min at 90°C in 10 mM EDTA pH 8.2 with 95% formamide will typically dissociate >96% of immobilized biotinylated DNA. Alternatively, boil the sample for 5 min in 0.1% SDS for protein dissociation. Please note that proteins will be denatured by such treatment and Dynabeads® Streptavidin can not be re-used. It has also been reported that the biotin-streptavidin interaction can be broken by a short incubation in nonionic water at a temperature above 70°C.
Magnetic separation and handling using Dynabeads® can easily be automated on a wide variety of liquid handling platforms. Dynabeads® MyOne™ Streptavidin T1 share similar properties to Dynabeads® M-280 Streptavidin but are smaller, making them ideal for automation applications due to their small size, low sedimentation rate and high magnetic mobility. Selected protocols are available at www.lifetechnologies.com/automation.