1. Overview

NHS-activated agarose magnetic beads (Magarose-NHS) provide a valuable method for affinity purification of antibodies, antigens, or other biomolecules by simply and efficiently covalently immobilizing proteins to magnetic bead carriers. Activated agarose magnetic beads contain N-hydroxysuccinimide (NHS) functional groups capable of reacting with primary amines on proteins or other molecules to form stable amide bonds. The coupling reaction was performed in an amine-free buffer at pH 7~9. Regardless of the molecular weight of the ligand or the efficiency of the isoelectric point (PI) coupling reaction, the efficiency is greater than 80%. Once the ligand is fixed, the prepared agarose beads can be used in multi-affinity purification procedures.

2. Product characteristics

Product Name	Magarose-NHS
Item number	PMAG017
Magnetic bead concentration	25% (v/v) in 100% isopropyl alcohol
ligand density	~ 50 μmol NHS/ml magnetic beads
Medium	6% cross-linked magnetic agarose
Particle size	20-45μm
Ligand binding	> 20 mg IgG/ml beads
Save	Store 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. Buffer

All water and buffers are filtered with 0.22 μm or 0.45 μm membranes.

(1) Washing Buffer A: 1 mM hydrochloric acid, cooled to 4ºC before use;

(2) Coupling Buffer A: 100 mM 2-morpholine ethysulfonic acid MES, pH 4.8 (for coupling of biomolecules with an isoelectric point of less than 7);

(3) Coupling Buffer B: 200 mM NaHCO₃, pH 8.3 (for coupling of biomolecules with isoelectric points greater than 7);

(4) Blocking Buffer: 3 M ethanolamine, pH 9.0;

(5) Storage Buffer: PBS buffer with 0.05% sodium azide or other buffers according to the customer's own needs.

 

2. Key points of the process

1) Among them, the magnetic bead washing should be strictly according to the instructions and used to use the cooled Washing Buffer A ((1)) for rapid washing to prevent the magnetic beads from hydrolyzing in the NHS group during the washing process;

2) In the process of protein coupling, the appropriate Coupling Buffer (mainly including Coupling Buffer A((2)) and Coupling B) should be determined through experiments ((3)), 50 mM boric acid solution pH 8.5, 100 mMPhosphate buffer,100mM NaCl, pH 7.4);

3) Once the appropriate coupling buffer has been determined, the appropriate conjugated protein concentration is determined based on this coupling buffer, because the higher the protein concentration, the greater the amount of protein conjugated to the beads (this is due to the competitive reaction between NHS group and protein coupling and NHS group hydrolysis itself). Of course, the performance and cost should be considered comprehensively, and some customers can meet the use needs by conjugating a small amount of protein, so it is possible to use a low concentration of protein, which can reduce the cost.

4) This step can be done with 3 M ethanolamine or Tris buffer (100 mM Tris-HCl, 150 mM NaCl, pH 8.0) for a minimum of 2 h or an additional step of BSA blocking if the background is still high after chemical blocking.

3. Protein coupling operation steps

The following procedure is illustrated using a 1.5 mL EP tube to take a 400 μL of magnetic bead sample. Users can adjust proportionally according to their own needs.

Protein solution preparation: Take an appropriate amount of protein to be conjugated and dissolve it with a Coupling Buffer to prepare a protein solution with a concentration of ≥3.0 mg/mL. For proteins that have been stored in the buffer, the substances containing primary amines in the original buffer need to be completely removed by dialysis or desalting, and then the protein solution with a concentration of ≥3.0 mg/mL is prepared with a Coupling Buffer, and the prepared protein solution is stored at 4°C for later use.

Note: (1) In order to achieve better performance, the protein concentration ≥ 3.0 mg/mL, so that the conjugation efficiency will be higher, and the cost and use requirements need to be comprehensively considered. (2) The protein solution should not contain primary amino acids, such as Tris, glycine, gelatin, BSA, etc.;

1) Take 400 μL of 25% magnetic bead suspension in a 1.5 mL EP tube. Magnetic separation to remove the supernatant.

Note: Before sampling, the magnetic beads should be repeatedly reversed and mixed evenly with a vortex oscillator to ensure the identity of the experiment.

2) Add 1 mL of Washing Buffer A ((1)) at 2~8°C to the centrifuge tube and vortex for 15 s to mix the beads evenly. The EP tube is placed in a magnetic separator rack to enrich the beads and remove the supernatant.

3) Add 200 μL of protein solution to the EP tube and vortex for 30 s to mix well.

Note: Add the protein solution immediately after washing the magnetic beads.

4) Vortex the EP tube for 15 s, place it on a vertical mixer, and mix at room temperature for 2~4 h. If the vertical mixing is not homogeneous, vortex the EP tube every 5 min for 15 s 30 min before the reaction. Thereafter, every 15 min, remove the EP tube and vortex for 15 s.

NOTE: Reaction can be left overnight at 4ºC if needed.

5) The magnetic beads are enriched with magnetic separation frames to preserve the supernatant. Add 1 mL of Blocking Buffer ((4)) to the EP tube, vortex for 30 s, place the EP tube in a magnetic separation rack, enrich the beads, and discard the supernatant.

Note: In addition to the 3 M ethanolamine provided in the kit, the Blocking Buffer ((4)) can also use other end-capping reagents such as 100 mM Tris-HCl, 150 mM NaCl, pH 8.0, etc.

6) Add 1 mL of Blocking Buffer ((4)) to the EP tube, vortex for 30 s, and place the EP tube in a vertical mixer for 2 h at room temperature. Place the EP tube in a magnetic separation rack, enrich the magnetic beads, and discard the supernatant.

7) Add 1 mL of ultrapure water to the EP tube, mix thoroughly, enrich the magnetic beads with a magnetic frame, and discard the supernatant.

8) Add 1 mL of Storage Buffer ((5)) (such as PBS buffer containing 0.05% sodium azide, or customers can choose a suitable storage solution according to their actual needs) to the EP tube, mix thoroughly, enrich the magnetic beads with a magnetic frame, and discard the supernatant. Repeat this operation 2 times.

9) Add 400 μL of Storage Buffer ((5)) to the EP tube, mix thoroughly, and store at 4ºC for later use.

NOTE: The magnetic bead concentration for the final conjugated protein is 25% (v/v).

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