M-270 Amine

Dynabeads® M-270 Amine are uniform, superparamagnetic beads composed of highly cross-linked polystyrene with magnetic material precipitated in pores evenly distributed throughout the beads. The beads are further coated with a hydrophilic layer of glycidyl ether which seals the iron oxide inside the beads, and the surface is activated with primary amino functionality on a short hydrophilic linker. The hydrophilic surface ensures low non-specific binding, excellent dispersion abilities and easy handling of the beads in a wide variety of buffers. The beads are sold in an aqueous suspension at a concentration of 2 x 10⁹ beads/ml (approx. 30 mg/ml).

Principle

Dynabeads® M-270 Amine act as a solid support in a wide variety of biomagnetic separations and manipulations. Their size makes them particularly suitable for protein isolation for sample preparation, bioassays, selection of affinity binders etc. Due to the gentle pull of the beads to the magnet, they can also be used for selection of fragile cells. For cell separation in general, the larger Dynabeads® (4.5 μm) are recommended.

Surface-reactive primary amino-groups allow immobilization of ligands such as proteins, peptides, carbohydrates or other target specific molecules through reductive amination of aldehyde or ketone groups without prior activation of the surface. Alternatively, ligands can be immobilized through amide-bond formation with carbodiimide-activated carboxylic acid groups. Bifunctional crosslinkers may be used to introduce other functional groups. Dynabeads® can be coated directly with peptides, carbohydrates, enzymes etc, for the isolation of targets such as hormones, receptors, lectins, disease markers, bacteriophages etc. Once coupled with ligand, the beads are added to the sample containing your target molecule. After a short incubation allowing affinity capture of the target, the beads are pulled to the side of the test tube by the use of a magnet (Dynal® MPC™), allowing aspiration of unbound material. The magnet facilitates washing and target concentration. Bead-bound targets can be used directly in bioassays, boiled in application buffer and analyzed on SDS-PAGE. Alternatively, the target molecule can be eluted off the beads with conventional elution methods (high salt, low pH etc.).

Instructions for Use

For ligand-coating of Dynabeads® M-270 Amine, 3 μg pure ligand per 10⁷ beads and a final concentration of 1-2 x 10⁹ beads per ml is recommended. Both ligand and bead concentration can be adjusted depending on the stock concentration, solubility and availability of your ligand. Higher bead concentrations increase the efficiency of immobilization, and higher ligand concentrations increase the loading on the beads. Calculation example: 100 μl beads = 2 x 10⁸ beads. Ligand requirement = 60 μg ligand (using 3 μg per 10⁷ beads). A ligand concentration of 1-10 mg/ml gives a volume of 6-60 μl. The total volume should be 100-200 μl, and hence the beads should be dissolved in 40-200 μl before addition of the ligand.

3.A.   Washing procedure

Washing is necessary to equilibrate the Dynabeads® M-270 Amine in an appropriate buffer.

1. Resuspend the beads well by pipetting and vortexing for 1-2 min. Avoid foaming.


2. Immediately pipette the volume to be used over to the desired test tube (e.g. 100 μl or 2 x 10⁸ beads).


3. Place the tube on a magnet for 4 min. Carefully pipette off the supernatant, leaving beads undisturbed.


4. Remove the test tube from the magnet and carefully resuspend the beads in the original sample volume, using a buffer of choice according to the preferred conjugation method.


5. Repeat steps 3 and 4 once.


6. Resuspend the washed beads in the same buffer to a volume that will give the recommended bead concentration after addition of protein solution (see calculation example above). The beads are now ready for coating.

3.B.   Coating procedures

Alternative immobilization-principles:

• Direct ligand coupling via aldehyde- or ketone groups by reductive amination (see section 3.B.1 below).
• Activation of the beads with a bi-functional crosslinker. An amine-reactive crosslinker will introduce a linker and a new functionality on the bead-surface for ligand coupling. The crosslinker should be chosen to give the preferred mode of immobilization of your specific ligand.
• Activation of the ligand with carbodiimide or crosslinker. A crosslinker may also be introduced on the ligand. The crosslinker should be chosen so that the free end of the crosslinker is amine reactive. Alternatively, carboxylic acid groups on the ligand may be activated with a carbodiimide and then reacted with the beads. This results in a direct amide bond formation between the beads and the ligand.

3.B.1   Coating procedure for conjugation of aldehyde or ketone containing molecules by reductive amination

Coupling of an aldehyde or ketone group in the ligand to amine on the bead surface can be achieved by Schiff base (imine) formation and reductive amination. Aldehyde groups can easily be prepared by sodium periodate oxidation of sugar residues in glycoproteins, or cleavage of carbon-carbon bonds with adjacent hydroxyl-groups in polysaccharides. The reductive amination is achieved by use of a reducing agent like cyanoborohydrid.

1. Dissolve the aldehyde containing ligand to a concentration of 1–10 mg/ml in an appropriate buffer (e.g. 0.1 M sodium phosphate buffer with 0.15 M NaCl, pH 7.4, or 0.1 M sodium borate pH 9.5 or 0.1 M sodium citrate pH 9.5). The same buffer should be used for pre-washing the beads.


2. Add the calculated amount of ligand solution to the homogenized bead-suspension. Vortex to ensure good mixing.


3. Add 1 μl of a solution containing 5 M cyanoborohydrid in 1 M NaOH (see section below) to each 100 μl of reaction mixture, vortex properly and incubate for 2 hours at room temperature with slow tilt rotation. Do not let the beads settle during the incubation period. Note that cyanoborohydrid is highly toxic, use a fume hood and avoid contact with skin.


4. After incubation, place the tube on the magnet for 4 min and remove supernatant.


5. Add 0.1 M ethanolamine adjusted to pH 7.4 to the same volume as previously used and incubate for 15 min at room temperature with slow tilt rotation. 6. Wash the coated beads as described in section 3.C below.

3.B.2  Activating with NHS-ester cross-linker

The most common kind of activating agents are NHS (N-hydroxy-succinimidyl)-esters. A large amount of different NHS-esters are commercially available for cross-linking. Depending on the nature of the crosslinker, this can react with chemical groups in the ligand to be immobilized. Reactivities include amine, sulfhydryl, carboxyl and hydroxyl as well as non-selective photoreaction. NHS-ester cross-linkers must normally be prepared just prior to use. Use a 10-fold molar excess compared to the amount of ligand to be immobilized.

1. Resuspend the beads in 0.1 M sodium phosphate buffer with 0.15 M NaCl, pH 7.4. Avoid amine containing buffers like Tris or glycine as they will compete with the NHS-ester reaction.


2. Dissolve the NHS-ester according to the manufacturer’s instruction and add the required volume to the bead-solution. Water-soluble NHS-esters may be added directly to the beads. Final volume should be equal to the bead-volume originally pipetted from the vial. Vortex to mix properly.


3. Incubate for 30 min at room temperature with slow tilt rotation.


4. After incubation, place the tube on the magnet for 4 min and remove the supernatant. Wash twice with the buffer above.


5. The beads are now activated and with a functional group according to the chosen cross-linker.

3.B.2.1  Coating after activation with NHSester cross-linker with amine-reactivity:

With homo-bifunctional cross-linkers a second NHSester group will react similarly with primary amines in the ligand, and hence these are normally used for immobilization of proteins or the N-terminal in peptides. After activating the beads as described in section 3.B.2 above:

1. Make a homogeneous suspension of the activated beads. If necessary, use the same buffer to adjust the volume to the required bead-concentration.


2. Add the calculated amount of ligand. Vortex to ensure good mixing.


3. Incubate for 30 min at room temperature or 2 hours at 4°C with slow tilt rotation.


4. After incubation, place the tube on the magnet for 4 min and remove the supernatant.


5. Add 0.05 M Tris pH 7 and incubate for 15 minutes at room temperature with slow tilt rotation, to quench non-reacted groups.


6. Wash the coated beads as described in section 3.C below.

3.B.2.2   Coating after activation with NHSester cross-linkers with sulfhydryl-reactivity:

Sulfhydryl reactive groups are often pyridyldithio or iodo/bromoacetyl. For oligonucleotides maleimide may also be used. Appropriate buffers and incubation conditions should be used to give reaction with sulfhydryl. The ligand must contain free sulfhydryl groups in order to be immobilized. After activating the beads as described in section 3.B.2 above:

1. Make a homogeneous suspension of the activated beads. Use a buffer according to the active group chosen (see table below). If necessary, use the same buffer to adjust the volume to the required bead/ligand concentration.


2. Add the calculated amount of free sulfhydryl-containing ligand. Vortex to ensure good mixing.


3. Incubate for the recommended time and temperature with slow tilt rotation.


4. After incubation, cysteine may be added to a final concentration of 5 mM to quench non-reacted groups. Incubate for 15 min at room temperature with slow tilt rotation.


5. Wash the coated beads as described in section 3.C below.

3.B.2.3  Coating after activation with NHSester cross-linkers with photoreactivity:
 

Photoreactive groups like hydroxylphenylazide, nitrophenylazide, phenylazide or perfluoroarylazide moiety may be used to immobilize ligands with amine groups. Activation with a cross-linker with photoreactive groups must be performed under dark-room conditions!

After activating the beads as described in section above:

1. Make a homogeneous suspension of the activated beads. If necessary, use the same buffer to adjust the volume to the required bead/ligand concentration.


2. Add the calculated amount of ligand. Vortex to ensure good mixing.


3. Irradiate with light with the appropriate wavelength using the recommended time and temperature conditions with slow tilt rotation. Note that the beads may quench the light, and hence the method may have to be optimized.


4. Wash the coated beads as described in section 3.C below.

Note:   The beads are compatible with organic solvents such as DMF, and both the activation and coupling steps described above can be performed in dry solvents. This will eliminate the competing hydrolysis reaction, thus higher yields may be achieved by using longer reaction times. Wash once in cold, purified water before transferring the beads form organic solvent to a salt-containing buffer.

3.B.3   Activation with cross-linkers with amine and carboxyl reactivity, for coating with carboxyl-containing ligand

Amine-groups on the surface of the beads and carboxyl-groups in the ligand may be linked together by use of EDC or EDC/NHS (or other carbodiimides) under the condition where no other primary aminogroups are present in the ligand. EDC reacts with carboxyl-groups to form an amine-reactive intermediate. This intermediate is unstable in aqueous solutions. To stabilize, NHS may be introduced.

1. Wash the beads with 0.1 M MES (2-[N-morpholino] ethane sulfonic acid), pH 4.5-5 (or 0.1 M MES, 0.5 M NaCl p  6.0).


2. Dissolve the ligand in the same buffer to a concentration of 1-10 mg/ml and add the recommended amount of ligand. Vortex to ensure good mixing.


3. Dissolve 10 mg EDC in 1 ml cold deionized water, (or dissolve 10 mg EDC and 15 mg NHS per ml). This must be done immediately prior to use.


4. Add 50-100 μl EDC (or EDC/NHS) solution for each mg ligand used. Vortex to mix properly.


5. Incubate for 2 hours at room temperature or 2 hours at 4°C with slow tilt rotation.


6. Add hydroxylamine (NH₂OH x HCl, MW 69.49) to a final concentration of 10 mM to quench the reaction, and incubate for 15 minutes at room temperature with slow tilt rotation.


7. Wash the coated beads as described in protocol 3.C below.

3.C.  Washing of coated beads

All immobilization procedures require washing of the coated beads to remove excess ligand.

1. Wash the coated beads a total of four times in PBS or similar. It is recommended to add BSA to 0.5% in the washing buffer for effective blocking of the surface (when this does not interfere with the downstream applications). Use 0.1% Tween-20 or Triton X-100 as an alternative or in addition to the BSA.


2. Resuspend the coated beads in PBS with 0.1% BSA and / or 0.01% Tween-20 / Triton X-100 to 1 x 109 bead  / ml.


3. For storage of the coated beads, add sodium azide to 0.02% and store at 2-8°C. Other bacteriostatic agents can be used. Coated beads can usually be stored for several months, depending on the stability of the immobilized ligand. If the coated beads are stored for more than two weeks, wash twice for five minutes with buffer suitable for your application prior to use.

3.D.   Isolation of target molecule

Efficient isolation of target molecules is dependent on the concentration of beads and target molecules, the ligand’s specific affinity and time.

1. Remove the storage buffer from 200 μl beads (2 x 10⁸ beads). Add 100-200 μl of the solution containing your target molecule. Excess target is needed if maximum binding is required (e.g., for a 100 kD protein, use 25 μg.)


2. Incubate with tilting/rotation for up to 1 hour to capture the target. Higher target and/or bead concentration increases the rate of binding.


3. Place the tube on the magnet for 4 min. to collect the beads at the tube wall (for viscous samples, increase the separation time). Pipette off the supernatant.


4. Wash the beads 3 times (using 1 ml PBS each time) by the use of the magnet.

3.E.  Target protein elution procedure

Conventional elution methods can be applied. Low pH (2.8-3.5), change in ionic strength, affinity elution, electrophoresis, polarity reducing agents, deforming eluants can be applied, or even boiling the beads in SDS-PAGE application buffer for direct protein characterization. The method of choice will depend on the affinity of the target molecule to the ligand coated onto the beads, stability of the target molecule as well as downstream applications and detection methods. Most proteins will be eluted off the beads at pH 3.1 following the procedure described below, but some protein functionality might be lost under such harsh conditions. If maintaining functionality of the target molecule is important, try milder elution conditions first, e.g. high salt (e.g. 2M NaI) or a stepwise elution reducing pH from 6 to 3. This approach is also recommended if the bead-bound ligand must remain functional to allow reuse of the beads.

1. Add 30 μl 0.1 M citrate (pH 3.1) to beads with immobilized target. Mix well by tilting and rotation for 2 minutes.


2. Place the test tube on a magnet and transfer the supernatant, containing purified target, to a clean tube.


3. Add 30 μl 0.1 M citrate (pH 3.1) to the beads to elute any remaining target. Mix well by tilting and rotation for 2 minutes.


4. Place the test tube on a magnet, pipette off the eluate and pool the supernatants containing pure target molecules. Total collected volume = 60 μl. To ensure reuse of the beads and functionality of the isolated target molecules, bring both beads and target back to physiological pH (7.4) immediately after elution

Typical bead-characteristics for any given lot of this product:
Diameter: 2.8 μm
Specific surface area: 2-5 m2/g
Active chemical functionality: 0.1 – 0.2 mmol/g
Surface charge: Positive at pH 2-9

4.2. Additional material needed

• Magnetic device (Dynal® MPC, Magnetic Particle Concentrator)
• Mixing/rotation device
• Test tubes, glassware and pipettes
• Antibody/other selecting molecule
• Buffers/solutions (see below)

4.3. Recommended buffers/solutions

Invitrogen Dynal® recommends the use of 0.1M Phosphate buffer pH 7.4, PBS and PBS Tween. Other buffers can also be used, but buffers containing amino groups (e.g., Tris) should not be used for coating of ligand. High pH and high temperature during coating procedures are optimal conditions for quick formation of chemical bonds. (The upper pH and temperature limit is determined by the ligand). The molarity of salt in the final coating solution should never be less than 0.05M.

0.1M Sodium phosphate buffer pH 7.4 with 0.15 M NaCl:
2.62 g NaH₂PO₄ x H₂O (MW 137.99)
14.42 g Na₂HPO₄ x 2H₂O (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.

0.1 M Sodium borate pH 9.5:
0.62 g H₃BO₃ (MW 61.83)
Dissolve in 90 ml distilled water, adjust to pH 9.5 and adjust to 100 ml.

0.05 M Sodium borate pH 8.3:
0.30 g H₃BO₃ (MW 60.83)
Dissolve in 90 ml distilled water, adjust to pH 8.3 and adjust to 100 ml.

0.1 M Sodium citrate pH 9.5.
2.94 g C6H5O7Na3 x 2H₂0 (MW 294)
Dissolve in 90 ml distilled water, adjust to pH 9.5 and adjust to 100 ml.

5 M Cyanoborohydrid in 1 M NaOH (highly toxic, avoid contact):
0.31 g NaCNBH₃ (MW 62.84)
Dissolve in 10 ml 1 M NaOH (40.0 g NaOH (MW 40.0) to 1 liter distilled water).

0.1 M Ethanolamine adjusted to pH 7.4:
0.6 ml C₂H₇NO (MW 61.08, d = 1.02 g/ml)
Dissolve in 90 ml distilled water, adjust to pH 7.4 and adjust to 100 ml.

0.05 M Tris pH 7.4:
0. 79 gTris HCl (MW 157.6)
Dissolve in 90 ml distilled water, adjust to pH 7.4 and adjust to 100 ml.

0.1 M MES pH 4.5-5:
2.13 g MES (MW 213.25)
Dissolve in 90 ml distilled water, adjust to pH 4.5- 5 and adjust to 100 ml.

0.1 M MES, 0.5 M NaCl pH 6:
2.13 g MES (MW 213.25)
2.93 g NaCl (MW 58.5)
Dissolve in 90 ml distilled water, adjust to pH 6.0 and adjust to 100 ml.

0.1 M Citrate pH 3.1:
2.10 g citric acid (C₆H₈O₇ x H₂O, MW 210.14)
Dissolve in 90 ml distilled water, adjust to pH 3.1 and adjust to 100 ml.

2 M NaI:
3 g NaI (MW 149.9) to 10 ml distilled water.

PBS pH 7.4:
0.26 g NaH₂PO₄ x H₂O (MW 137.99)
1.44 g Na₂HPO₄ x 2H₂O (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.

PBS with 0.1% (w/v) BSA/HSA/skimmed milk:
Include 0.1% (w/v) BSA/HSA/skimmed milk
(0.1g) in 100 ml PBS (above).
PBS/Tween 20/Triton X:
Include 0.5-1.0 % (w/v) Tween 20/Triton X (50- 100 mg) in 100 ml PBS (above).