1. Overview

The Amino-Activated Agarose Magnetic Bead (Magarose-NH₂) is a 6% cross-linked magnetic agarose microsphere with a reactive primary amine, a molecule covalently coupling containing a carboxyl group (-COOH) at the end of a long spacer arm for affinity purification. Gels are ideal for immobilized peptides for affinity purification of antibodies or other binding gametes.

Although other amine-reactive methods immobilize molecules on amino-activated agarose beads, carbodiimide (EDC) is unique among crosslinkers, enabling coupling by -COOH. In near-neutral to acidic pH buffers, EDCs efficiently react with carboxylates to form intermediate esters that react with primary amines on nucleophiles such as amino-activated agarose beads. In most affinity purification methods, the amide bonds created between the molecule and the agarose gel carrier are stable and leak-proof.

Amino-activated agarose beads are also suitable for immobilization of oligonucleotides by 5'-phosphate groups. In the presence of imidazoles, EDCs react effectively with the phosphate group in much the same way as carboxylates.

2. Product characteristics

Product Name	Magarose-NH2
Item number	PMAG018，PMAG019
Magnetic bead concentration	25% (v/v) in 0.02% sodium azide
ligand density	~ 60 μmol NH2/ml beads
Medium	6% cross-linked magnetic agarose
Particle size	20-45μm
Ligand binding	> 20 mg IgG/ml beads
Save	Transported at room temperature and stored at 2~8°C for one year

Note: The amount of magnetoglobin binding is related to the characteristics of the target protein, and is only used as a reference value.

3. How to use

1. Important Product Information

• When using EDC, amino beads will be coupled to the side chain-COOH of the C-terminus, aspartic acid, or glutamate residues. Because peptides also contain primary amines (side chains of N-terminus and lysine residues), coupling using EDCs will lead to the polymerization of peptides as well as their immobilization to the gel support. Usually this polymerization is not harmful to subsequent affinity purification methods.

• When attaching water-insoluble peptides or other molecules, use solvents miscible with water, such as ethanol, methanol, DMSO, or DMF. The peptide is first dissolved in a water-miscible solvent, and then the solution is added to the conjugation buffer. Up to 50% of the organic solvent concentration in the conjugation reaction is compatible unless the peptide is known to denature at that concentration.

• Traditionally, MES buffer has been used for EDC reactions because it does not contain competitive amines or phosphates and effectively maintains optimal conjugated acid conditions. However, other buffers can be substituted if needed, and coupling will be effectively performed up to pH 7.2. Although phosphate buffer can react with EDC, it reduces the coupling efficiency. Acetate, Tris, and glycine buffers react with EDCs or O-acylisourea intermediates and are not suitable conjugation buffers. Also, avoid buffers containing mercaptans, which irreversibly bind to and inactivate EDCs. Halides such as iodide, chloride, and bromide have little effect on EDCs with pH between 5 and 7.

• The EDC/Magarose-NH2 method can also be used to fix oligonucleotides through the 5'-phosphate group.

 

2. Use EDC and Magarose-NH₂peptide immobilization method

Note: This method uses 2 mL of magnetic beads Magarose-NH₂ (8 mL of slurry) conjugated to 1-10 mg of peptides. For other volumes and peptide amounts, please adjust the program accordingly.

 

A. Materials needed:

· Conjugation buffer: 0.1M MES buffer [2-(N-morpholine)ethylene acid], 0.9% NaCl, pH 4.7

• Washing Solution: 1M NaCl

• EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl]

 

B. Reagent preparation

Dissolve 1-10 mg of peptide in 2 mL of conjugation buffer. To estimate the coupling efficiency after peptide conjugation, the absorbance of the sample at 280 nm was measured. (This method assumes that the peptide is absorbed at 280 nm and can use other wavelengths to detect the presence of the peptide with a separate coupling buffer).

 

C. Magnetic bead preparation

Carefully transfer the 8 mL magnetic beads into the glass vial, magnetically separate, and remove the supernatant. Add 10 ml of coupling buffer to equilibrate the beads. Magnetic separation, remove supernatant, and set aside magnetic beads.

 

D. Peptide coupling

1) Equilibrate the EDC vial to room temperature before opening to avoid moisture condensation into the vial.

2) Add 2 mL of the prepared peptide sample to the magnetic beads and gently invert the mixed/magnetic bead slurry for a few minutes.

3) Add 0.5 mL of conjugation buffer to 60 mg EDC.

Note: EDCs are sensitive to moisture and hydrolyze rapidly when dissolved in aqueous buffer. For best results, seal the dry powder reagent in a desiccant at -20°C in its original vial. Quickly dissolve the required amount of reagent immediately before use and discard any unused solution.

4) Immediately after the EDC is dissolved, add 0.5 mLEDC solution to the sample/magnetic bead slurry from step 2. Gently mix the slurry at room temperature for 3 hours.

5) Magnetic separation, removal of supernatant. Add 2 mL of wash, magnetically separate, and collect the supernatant. The two parts of the supernatant were mixed and used to determine the coupling efficiency.

NOTE: The collected sample (~4 mL) contains unbound peptides. To measure the conjugation efficiency, compare the absorbance of the solution with the starting peptide sample, considering the 2-fold dilution effect.

6) Add 2mL of wash solution to the magnetic beads and wash three times.

7) Magnetically separated magnetic beads should be balanced in phosphate-buffered saline (PBS) or other suitable buffer containing 0.05% sodium azide and stored at 4°C.

 

3. Method of fixing nucleic acids or oligonucleotides by means of a 5'-phosphate group

Note: Use 1 μL of gel (4 μL of mixed slurry) for every 1 μg of oligonucleotide to be conjugated.

A. Materials needed:

• 0.1 M imidasole, pH 6

• Ultra-pure DNase and/or RNase water

 

B. Nucleic acid or oligonucleotide conjugation

1) Mix Magarose-NH₂ slurry to obtain a homogeneous magnetic bead suspension. Use a pipette to transfer the appropriate amount of magnetic beads to a microcentrifuge tube.

2) Magnetically separate and discard the supernatant.

3) Wash the beads with ultrapure water with 2 beads sedimentation volume 3-5 times, magnetically separate and remove the supernatant each time.

4) For magnetic beads per microliter used, dissolve up to 10 μg of DNA or RNA in 1 μL of 0.1 M imidazole (pH6).

5) Add the nucleic acid solution to the gel and mix well.

6) Weigh 1 mg of EDC and dissolve in 67 μL of 0.1 M imidazole (pH 6).

7) For each microliter of magnetic beads, add 2 μL of EDC solution.

8) Shake or mix the reaction at room temperature for 3 hours.

9) Magnetically separate and remove supernatant containing unbound nucleic acids.

10) Wash the beads 3-5 times with 2 volumes of water or an appropriate wash solution (e.g., Tris-EDTA), centrifuge and discard the supernatant each time.

Magnetically separated magnetic beads are stored at 4°C in phosphate-buffered saline (PBS) or other suitable bourer containing 0.05% sodium azide.