Introduction

Dynabeads® MyOne™ Carboxylic Acid are uniform, monosized superparamagnetic 1 μm in diameter beads, which are composed of highly cross-linked polystyrene with evenly distributed magnetic material. The beads are further coated with a hydrophilic layer of glycidyl ether, concealing the iron oxide inside them. Carboxylic acid groups are then introduced on the surface of the beads. The small 1 μm Dynabeads® MyOne™ Carboxylic Acid have hydrophilic surface, which ensures low non-specific binding, excellent dispersion abilities and easy handling in a wide variety of buffers. These beads have also a high magnetic mobility combined with low sedimentation rate making them ideal for automated assays. Dynabeads® MyOne™ Carboxylic Acid are supplied in an aqueous suspension.

Principle

Dynabeads® MyOne™ Carboxylic Acid provides an excellent solid support for a wide range of biomagnetic separations, molecular manipulations and affinity isolations. Their size makes them particularly suitable for sample preparation and handling of nucleic acids and proteins. A carbodiimide is utilized to activate Dynabeads® MyOne™ Carboxylic Acid for amide bonding with primary amines.

The mechanism and optimization of carbodiimide mediated amide bond formation is extensively discussed in the literature (1,2,3,4). Bifunctional cross-linkers may be used to introduce other functional groups like thiol, amine, maleimide etc, onto the surface of the beads. If the ligand to be bound does not contain a primary amino function, this can be introduced by e.g. using 5'-amino modified oligonucleotides. Please note that other amino-groups in the oligonucleotide might to some degree react with the carboxylic acid groups on the beads, resulting in coupling via the internal bases.

Once coupled with your ligand, the Dynabeads® can be added to a cell lysate or other suspensions containing your target molecule. After a short incubation allowing affinity capture of the target, the Dynabeads are pulled to the side of the test-tube by the use of a magnet (Dynal® MPC™) allowing aspiration of unbound material. Furthermore, the magnetic separation facilitates washing and concentration of the isolated target bound to the beads. Dynabeads® do not inhibit enzymatic activity, and can be included directly in downstream analysis of the bead-bound target molecule. Alternatively, the target molecule can be eluted off the Dynabeads® with conventional elution methods.

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Protocol

Instructions for Use

For coating of a ligand to Dynabeads® MyOne™ Carboxylic Acid, Invitrogen Dynal® recommends to use 50 μg pure protein or ~1800 pmol oligonucleotides/peptides per mg Dynabeads® and a final concentration of 10-30 mg beads per ml during incubation.

Oligonucleotides can be coupled to Dynabeads® MyOne™ Carboxylic Acid via the 5' or 3' after amino (NH2) modification by use of EDC to establish an amide bond. The suggested protocols described in the sections below, illustrate an example using 3 mg of Dynabeads® , but should be scaled up or down to suit specific needs. It is recommended that the protocols are optimized to meet your requirements (e.g. sample volume, concentration of ligand/beads/EDC, MES buffer volume and pH).

Calculations:

The concentration of the supplied beads is 10mg/ml.  3mg beads = 300 μl.

Protein concentration is in this example set to 2.5 mg/ml. NH2;-oligo concentration is 90 μmol/ml.

Given that 50 μg protein or 1800 pmol oligo is required per mg Dynabeads® , the required amount of protein or oligo in this case is 150 μg and 5400 pmol respectively. This corresponds to a volume of 60 μl ligand.

The required amount of EDC varies depending on the performed coating procedure. The final sample volume should be 100 μl to meet the recommendation of final Dynabeads® concentration of 30 mg/ml. In brief, according to the calculation above you require:

300 μl of washed Dynabeads® (see the washing step under each protocol)
60 μl ligand.
MES Buffer to adjust the final volume to 100 μl.

Activation and Coupling of Ligand

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Formation of an amide bond between a primary amino group of the ligand and the carboxylic acid groups on the surface of the Dynabeads® is mediated by carbodiimide activation. The intermediate product of the reaction between the carboxylic acid and the carbodiimide is very labile and will hydrolyze quickly. To get the desired immobilization of the ligand it is therefore important to have the ligand immediately available. Alternatively, the activated Dynabeads® can be captured as a less labile intermediate, like an N-hydroxyl succinimide ester (use NHS, MW 115.1 or sulfo-NHS, MW 217.1), and then react with the ligand over a longer period. There are several alternative protocols for carbodiimide-mediated immobilization of ligand by amide bond formation:

One-step protocol (see protocol below):

 

  1. Mix ligand and Dynabeads® .

  2. Add carbodiimide and incubate the reaction mixture.

  3. Wash the Dynabeads® and resuspend in buffer.


Two-step protocol (see protocol  below):

  1. Add carbodiimide to the Dynabeads® .

  2. After 30 minutes incubation, quickly wash the Dynabeads® in cold water followed by cold buffer.

  3. Add the ligand to the Dynabeads® and incubate further.

  4. Wash the Dynabeads® and resuspend in buffer.

 

Two-step protocol with NHS (see protocol below):                                                                                                                                                                                                                 

 

  1. Add a mixture of carbodiimide and N-hydroxy succinimide to the Dynabeads® .

  2. After 30 minutes incubation, wash the Dynabeads® to remove excess carbodiimide.

  3. Add the ligand to the Dynabeads® and incubate further.

  4. Wash the Dynabeads® and resuspend in buffer.


The one-step protocol is recommended when using ligands that do not contain carboxylic acid groups (e.g. oligonucleotides). If the ligand does contain carboxylic acid groups, these may react with the carbodiimide and cause polymerization of the ligand. Since this method is less laborious and generally gives higher yields, it may however still be advantageous to use this method if it is possible to add ligand in excess to compensate for the loss due to polymerization.

The two-step protocol is preferred when the ligand contains carboxylic acid groups and you have limited amounts of the ligand available. The two-step protocol without NHS requires a very fast wash of the beads in cold buffer after the activation. The two-step protocol with NHS should be used if the ligand is in an alkaline buffer or a buffer with high phosphate concentration.

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, MW 191.7) is the carbodiimide most commonly used. It is very soluble in water and relatively stable as an aqueous solution. For some applications, other carbodiimides may slightly improve the results. As an example: When coupling IgG or IgM, without using NHS, the more hydrophobic carbodiimide N-cyclohexyl-N'-(2-morpholinoethyl) carbodiimide methyl-p-toluensulfonate (CMC, MW 423.6) generally improves yield and orientation of the immobilized antibody.

One-Step Coating Procedure

  1. Wash Dynabeads® MyOne™ Carboxylic Acid twice with 25mM MES, pH 6, using the equal volume of Dynabeads® (300 μl) pipetted out of the vial, for ten minutes with good mixing (en-over-end or similar).

  2. Add the required amount of ligand in 25 mM MES, pH 6 (60 μl) to the washed Dynabeads® . Mix well and incubate with slow tilt rotation at room temperature for 30 minutes.

  3. Immediately before use dissolve EDC in cold 25 mM MES, pH 6 to a concentration of 10 mg/ml.

  4. Add 30 μl EDC solution (0.3 mg) to the Dynabeads® /ligand suspension. Mix well.

  5. Add 10 μl of 25 mM MES, pH 6 to final volume of 100 μl.

  6. Incubate for 2 hours or longer (over night) at 4°C with slow tilt rotation.

  7. Wash the coated Dynabeads® as described below
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Two-Step Coating Procedure (without NHS)

            I)    Activation with EDC

  1. Wash Dynabeads® MyOne™ Carboxylic Acid twice with 0.01 M NaOH, using the equal volume of Dynabeads® (300 μl) pipetted out of the vial, for ten minutes with good mixing (en-over-end or similar).

  2. Wash three times with 300 μl de-ionized water in the same manner. Remove excess liquid.

  3. Dissolve the EDC in cold, de-ionized water to 20 mg/ml. Add 200 μl of EDC-solution to the Dynabeads® . Vortex to mix properly.

  4. Incubate for 30 minutes at 2-8°C with slow tilt rotation.

  5. After incubation, place the tube on the magnet for 4 minutes and remove the supernatant. Wash once with 300 μl cold, de-ionized water and once with 300 μl 25 mM MES, ph 6, as quickly as possible to avoid hydrolysis of the activated carboxylic acid groups. The Dynabeads® are now activated and ready for coating with a ligand containing primary amine groups. Activated beads cannot be stored and you should proceed directly to the next step:


            II) Immobilization of ligand after activation

  1. Remove the wash solution used in step 5 above. Add the required amount of ligand in 25 mM MES, pH 6 (60 μl) to activated Dynabeads® .

  2. Add 40 μl of 25 mM MES, pH 6 to final volume of 100 μl. Vortex to ensure good mixing.

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

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

  5. Wash the coated Dynabeads® as described below


Two-Step Coating Procedure using NHS

            I) Activation with EDC and NHS

  1. Wash Dynabeads® MyOne™ Carboxylic Acid twice with 25 mM MES, pH 6, using the equal volume of Dynabeads® (300 μl) pipetted out of the vial, for ten minutes with good mixing (en-over-end or similar).

  2. Immediately before use dissolve EDC in cold 25 mM MES, pH 6 to a concentration of 50 mg/ml.

  3. Similarly, prepare a 50 mg/ml solution of NHS in 25 mM MES, pH 6.

  4. Add 50 μl of EDC solution and 50 μl of NHS solution to the washed Dynabeads® . Mix well and incubate with slow tilt rotation at room temperature for 30 minutes.

  5. After incubation, place the tube on the magnet for 4 minutes and remove the supernatant. Wash twice with 300 μl of 25 mM MES, pH 6. The Dynabeads® are now activated and ready for coating with a ligand containing primary amine groups.


            II) Immobilization of ligand after activation

  1. Add the required amount of ligand in 25 mM MES, pH 6 (60 μl) to activated Dynabeads® .

  2. Add 40 μl 25 mM MES, pH 6 to final volume of 100 μl. Vortex to ensure good mixing.

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

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

  5. Wash the coated Dynabeads® as described below


Washing of Coated Beads

All immobilization procedures require washing of the coated Dynabeads® to remove excess ligand and to block un-reacted surface.

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NOTE:   In order to quench the non reacted activated carboxylic acid groups, incubate the Dynabeads® coated with ligand with either 50 mM Tris pH 7.4 for 15 minutes or 50 mM ethanolamine in PBS pH 8.0 for 60 minutes, both at room temperature with slow tilt rotation.

  1. Wash the coated Dynabeads® a total of four times with 300 μl PBS or 50 mM Tris. Blocking protein like BSA or skimmed milk powder may be added to a concentration of 0.1 - 0.5 % when it does not interfere with downstream applications of the Dynabeads®. Also 0.1% Tween- 20 or Triton X100 can be added during washes, if necessary.

  2. Resuspend the coated Dynabeads® to the desired concentration in PBS or a Tris storage buffer. The Dynabeads® are now ready for use. Store the coated Dynabeads® at 2-8°C. Addition of 0.1 - 0.5% protein (BSA) and/or 0.01-0.1% Tween-20 or Triton X-100 is recommended to stabilize the immobilized ligand and increase the ease of handling. Coated Dynabead® s can usually be stored for several months at this temperature, depending on the stability of the immobilized ligand. A final concentration of 0.02% (w/v) sodium azide (NaN3) may be added as a bacteriostatic agent. If the coated Dynabeads® are stored for more than two weeks, they should be washed twice for five minutes with a buffer suitable for the application prior to use.


Isolation of Target Molecule

Efficient isolation of target molecules using Dynabeads® is dependent on the bead-concentration, target molecule concentration, the ligand's affinity for the target molecule and the specific binding kinetics involved.

The concentration of required Dynabeads® will depend on the size of your specific molecule. Also the salt-concentration and pH of the chosen binding, washing and elution buffers can be varied depending on the type of molecule to be immobilized. Similarly, the selected buffer used in the downstream application should be optimized for the specific application. The small size of the Dynabeads® MyOne™ Carboxylic Acid presents a high surface area per mg beads and a corresponding high capacity for the target molecule. The effective binding capacity  will depend on the size of the specific molecules to be immobilized.

As the Dynabeads® MyOne™ Carboxylic Acid will not inhibit enzymatic activity, bead-bound material can be used directly in downstream analysis. Alternatively, the target molecule can be eluted off the Dynabeads® following conventional elution methods.

Additional Material Needed

  • Magnetic device (Dynal® MPC™, Magnetic Particle Concentrator)
  • Mixing/rotation device (Dynal® Sample Mixer or similar)
  • Test tubes, glassware and pipettes
  • Ligands
  • Buffers/solutions


Recommended Buffers/Solutions

Listed below are some recommended buffers for use with Dynabeads® MyOne™ Carboxylic Acid:

0.01 M NaOH:

0.4 g NaOH (MW 40.0) dissolved in 1 litre distilled water.

25 mM MES, pH 6:

0.53 g MES (2-[N-morpholino]ethane sulfonic acid, MW 213.25). Dissolve in 90 ml distilled water, adjust to pH 6 and adjust to 100 ml.

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

PBS pH 7.4 (phosphate buffered saline):
0.26 g NaH2PO4 x H2O (MW 137.99). 1.44 g Na2 HPO4 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 litre.

PBS with 0.1% (w/v) BSA/HSA/skimmed milk:

Include 0.1% (w/v) BSA/HSA/skimmed milk (0.1 g) 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). If a preservative is needed for storage of coated Dynabeads® , a final concentration of 0.02% (w/v) sodium azide (NaN3) may be added to the storage buffer. This preservative is cytotoxic and must be carefully removed before use by washing. Required safety precautions must be followed when handling this material.

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General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Product Characteristics

Typical characteristics for any given batch of this product:
Bead diameter: 1.05 μm
Active chemical functionality: 0.6 mmol/g beads
Concentration: 10 mg/ml (7-12 x 10 9 beads/ml)
Density: 1.8 g/cm³
Certificate of Analysis (CoA) and Material Safety Data Sheet (MSDS) are available upon request.

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References

  1. Nakajima N and Ikade Y, "Mechanism of Amide Formation by Carbodiimide for Bioconjugation in Aqueous Media", Bioconjugate Chem. 1995, 6(1),123-130.

  2. Gilles MA, Hudson AQ and Borders CL Jr, "Stability of water-soluble carbodiimides in aqueous solution", Anal Biochem. 1990 Feb 1;184(2):244-248.

  3. Sehgal D and Vijay IK, "A method for the high efficiency of water-soluble carbodiimide-mediated amidation", Anal Biochem. 1994 Apr;218 (1):87-91.

  4. Szajani B et al, "Effects of carbodiimide structure on the immobilization of enzymes", Appl Biochem Biotechnol. 1991 Aug;30(2):225-231.
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DN101263_650.11_12_Rev006_Spec-05779.indd     5-May-2010