Introduction

Intended Use

Dynabeads® Streptavidin are ideal for numerous applications, including purification of proteins, nucleic acids purification, protein interaction studies, immunoprecipitation, immunoassays, phage display, biopanning, drug screening and cell isolation.

Guidelines for Use

All Dynabeads® Streptavidin can be used with biotinylated molecules. Some beads are more suitable for particular applications due to their characteristics. Dynabeads® M-280 Streptavidin and Dynabeads® MyOne™ Streptavidin T1 are commonly used for protein and nucleic acids applications. Dynabeads® M-270 Streptavidin and Dynabeads® MyOne Streptavidin C1 are preferred for nucleic acid diagnostics, specifically with samples with a high chaotropic salt concentration, immunoassays involving small biotinylated antigens and applications that are not compatible with BSA (these beads are not blocked with BSA). MyOne Dynabeads® offer increased binding capacity and slower sedimentation rate, making them ideal for automated applications and for when larger amounts of biotinylated ligand, or their specific target, need to be isolated.

Description of Materials

Dynabeads® Streptavidin are uniform, superparamagnetic beads 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. These beads give simple and stable binding of biotinylated molecules such as small molecules, peptides, proteins, antibodies, sugars, lectins, oligonucleotides, DNA/RNA etc.

Materials Supplied

Table 1: 
Materials supplied in the Dynabeads® Streptavidin Trial Kit. Sodium azide (NaN3) is used as a preservative. Abbreviations: Volume (V) Phosphate buffered saline (PBS)

Dynabeads® V [ml] Concentration [mg/ml] Supplied in Bead diameter [μm]
M-280 Streptavidin 1 10 6-7x108 beads/ml) PBS, pH 7.4 / 0.1% BSA / 0.02% NaN3 2.8
M-270 Streptavidin 1 10 (6-7x108 beads/ml) PBS, pH 7.4, / 0.09% NaN3 2.8
MyOne™ Streptavidin C1 1 10 (7-12x109 beads/ml) PBS, pH 7.4 / 0.01% Tween®-20 / 0.09% NaN3 1.0
MyOne™ Streptavidin T1 1 10 (7-12 x 109 beads/ml) PBS, pH 7.4 / 0.1% BSA / 0.02% NaN3 1.0

Additional Materials Required

  • Magnet (Dynal® MPC™) for manual or automated protocols. See recommendations.
  • Mixing device with tilting and rotation.
  • Buffers and Solutions (see Table 2).
  • Biotinylated compounds. For advice on biotinylation, see dynal. Section 4.2 of the handbook (http://probes.invitrogen.com/handbook) gives a guide available biotinylation reagents.

Table 2:
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):

10 mM Tris-HCl (pH 7.5)

1 mM EDTA

2 M NaCl
Solution A: DEPC-treated 0.1 M NaOH,
DEPC-treated 0.05 M NaCl

Solution B: DEPC-treated 0.1 M NaCl
PBS buffer pH 7.4 These buffers can also be used for your application if needed:

 PBS/BSA (PBS, pH 7.4 containing 0.1% (w/v) BSA) PBST (PBS, pH 7.4 containing 0.01% (v/v) Tween® 20)

The salt concentration and pH (typically 5-9) of the chosen binding/washing buffers can be varied depending
on the type of molecule to be immobilized. Beads with immobilized molecules are stable in common buffers.

DEPC-treatment:

Add DEPC to a final concentration of 0.1% (1ml/L) to Solution A or B. Shake vigorously, incubate for 1 hour at room temperature. Ready to use after autoclaving.

Recipe for PBS buffer pH 7.4:

0.26 g NaH2PO4 x H2O (MW 137.99)
1.44 g Na2HPO4 x H2O (MW 177.99)
8.78 g NaCl (MW 58.5)

Dissolve in 900 ml distilled water, adjust pH if necessary and adjust to 1 liter.

Principle

Add Dynabeads® to a sample containing biotinylated molecules. During a short incubation, the biotinylated molecule will bind to the beads. Separate the molecule-bead complex with a magnet. Capture, washing and detection can be optimized for manual or automated use. With indirect capture, the biotinylated molecule is mixed with the sample to capture the molecule-target complex before adding Dynabeads® . Indirect target capture can be advantageous 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.

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Protocols

Critical Notes

  • In the protocols we recommend keeping the tube on the magnet for up to 2 mins to ensure that all the beads are collected on the tube wall. For non-viscous samples, separation is often complete in under 1 min, once you can see the beads collected.
  • If you do not need to remove preservatives or change buffers you can omit Washing Procedure.
  • For diluted sample or large sample volumes, increase the incubation time or isolate in smaller batches using the same beads in each batch.
  • Use a mixer to tilt/rotate the tubes so Dynabeads® do not settle at the tube bottom.
  • Avoid air bubbles during pipetting.
  • Free biotin or biotinylated primer in the sample will reduce the binding capacity of the beads. A disposable separation column or a spin column will remove unincorporated biotin. Run the PCR with limiting concentrations of biotinylated primer, or remove free biotinylated primer by ultrafiltration, microdialysis or other clean-up protocols.

Immobilization Procedure

Bead Preparation

  1. Resuspend the beads in the original vial by rotation or vortexing.
  2. Calculate the amount of beads required based on their binding capacity, see table 3, and transfer the beads to a new tube.
  3. Wash Dynabeads® to remove preservatives.

  4. Recommended washing buffers:

    • nucleic acid applications: 1x B&W Buffer
    • antibody/protein applications: PBS, pH 7.4

    Note:  
    For many applications it can be an advantage to add a detergent e.g. 0.01-0.1% Tween® 20 to the washing/binding buffers to reduce non-specific binding.


    Washing Procedure

     
  5. Place the tube containing the beads on a magnet for 1-2 mins.
  6. Remove the supernatant by aspiration with a pipette while the tube is on the magnet.
  7. Remove the tube from the magnet.
  8. Add washing buffer along the inside of the tube where the beads are collected and resuspend
    (same volume of washing buffer as the initial volume of Dynabeads® taken from the vial or larger).
  9. Repeat steps 4 to 7 twice, for a total of 3 washes.

    If using Dynabeads® for RNA Manipulation:

    As Dynabeads® Streptavidin are NOT supplied in RNase-free solutions, perform the following steps after washing for RNA applications:

  10. Wash the beads twice in Solution A for 2 mins. Use the same volume of beads as recommended in step 7.
  11. Wash the beads once in Solution B. Use the same volume of beads as in step 9.
  12. Resuspend the beads in Solution B.

    The beads are now ready to be coated with the biotinylated molecule of your choice.

    General Immobilization Protocol

    Wash the Dynabeads® before use.
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    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 mins 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.

    Immobilization of 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 H2O to dilute the NaCl concentration in the 2x B&W Buffer from 2M to 1 M for optimal binding.
    3. Incubate for 15 mins at room temperature using gentle rotation. Incubation time depends on the nucleic acid length: short oligonucleotides (< 30 bases) require max. 10 mins. DNA fragments up to 1 kb require 15 mins.
    4. Separate the biotinylated DNA/RNA coated beads with a magnet for 2-3 mins.
    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.

    Immobilization of Antibodies/Proteins

    1. Incubate the beads and biotinylated antibodies in PBS for 30 mins at room temperature using gentle rotation.
    2. Separate the antibody-coated beads with a magnet for 2–3 mins.
    3. Wash the coated beads 4–5 times in PBS containing 0.1% BSA.
    4. Resuspend to the desired concentration for your application.


    Release of Immobilized Biotinylated Molecules

    The biotin-streptavidin bond is broken by harsh conditions. 5 mins incubation at 65°C or 2 mins at 90°C in 10 mM EDTA pH 8.2 with 95% formamide will typically dissociate >96% of immobilized biotinylated DNA. Alternatively, boi  the sample for 5 mins in 0.1% SDS for dissociation. Please note that proteins will be denatured by such treatmen  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 aqueous solution at temperature above 70°C.

    Immunoassay Strategies

    Due to their high surface area per weight, uniformity, excellent batch reproducibility and ease of adaptation to automated processes, Dynabeads® have become the solid phase of choice for developing immunoassays

    Automation

    Magnetic separation and handling using Dynabeads® can easily be automated on a wide variety of liquid handling platforms. Dynabeads® MyOne are ideal for automation applications due to the small size, low sedimentation rate and high magnetic mobility.

    Technical Information

    Binding capacity

    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.

    • Optimize the quantity of beads used for each individual application by titration.
    • Use up to two-fold excess of the binding capacity of the biotinylated molecule to saturate streptavidin.
    • Binding efficiency can be determined by comparing molecule concentration before and after coupling.

    Table 3:   Typical binding capacities for one mg of Dynabeads
    ® .


    Dynabeads® M-280 Streptavidin M-270 Streptavidin MyOne™ Streptavidin C1 MyOne™ Streptavidin T1
    Free Biotin [pmol]650 – 900≥ 950≥ 25001100-1700
    Biotinylated peptides [pmol]~ 200~ 200~ 400~ 400
    Biotinylated antibody [μg]up to 10up to 10up to 20up to 20
    ds DNA [μg] *)~ 10 ~ 10 ~ 20 ~ 20
    ds DNA [μg] *)~ 200~ 200~ 500 ~ 400

    *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.

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Reference

  1. Holmberg et al. (2005) The biotin-Streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 26, 501-510.
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