Fluorescein Isothiocyanate (FITC) - conjugated Antibodies

Intended Use
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.

Principle
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.

Materials Supplied

• 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:

• 2 ml (Cat.no. 112.05D)
• 10 ml (Cat. no. 112.06D)
• 100 ml (Cat. no. 602.10).

Additional Materials Required
Magnet for manual or automated protocols.

Table 1: Recommended buffers and solutions

Additional Material Needed

• Magnetic device (Dynal® MPC, Magnetic Particle Concentrator).
• Mixing/rotation device
• Vortex mixer
• Test tubes and pipettes
• Buffers/solutions
• Primary antibody

Recommended Buffers/Solutions

• All reagents used should be analytical grade. Isotonic (PBS) pH 7.2-7.6 Invitrogen Dynal® recommends the following PBS (pH 7.4):
• NaH₂PO₄ x H₂O 0.16 g
• Na₂HPO₄ x 2 H₂O 0.98 g
• NaCl 8.10 g
• Distilled water to 1 litre
• 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

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.

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.
• For diluted samples, 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 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.

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.

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.

Washing Procedure


4. Place the tube containing the beads on a magnet for 1-2 mins.
5. Remove the supernatant by aspiration with a pipette while the tube is on the magnet.
6. Remove the tube from the magnet.
7. 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).
8. 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:
9. Wash the beads twice in Solution A for 2 mins. Use the same volume of beads as recommended in step 7.
10. Wash the beads once in Solution B. Use the same volume of beads as in step 9.
11. 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.

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, boil  the sample for 5 mins 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 non-ionic water at a temperature above 70°C (ref 1).

Immunoassay Strategies

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

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.

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