Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
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.
3.B. Coating procedures
Alternative immobilization-principles:
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.
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.
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:
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:
3.B.2.3 Coating after activation with NHSester cross-linkers with photoreactivity:
SH-reactive group | Recommended buffer | Incubation conditions |
Maleimide | 0.1 M sodium phosphate pH 6.5-7.5 | 2 hours room temperature or 4 hours at 4°C |
Iodo/Bromoacetyl | 0.05 M sodium borate pH 8.3 | 1 hour at room temperature Protect from light |
Pyridyldithio | Phosphate buffered saline (PBS) pH 7.5 | Over night at room temperature |
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:
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.
3.C. Washing of coated beads
All immobilization procedures require washing of the coated beads to remove excess ligand.
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.
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.
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
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).