1. Overview

Epoxy activated agarose-epoxy is a newly developed activated magnetic bead by PuriMag, which uses 6% highly cross-linked agarose gel with an average particle size of 20-45μm, and uses macromolecular sugar linking branches on the surface, so that it has a higher specific surface area and better biocompatibility. Due to the large specific surface area, the equilibration and elution time is also shorter. It is grafted Even if it purifies large molecules such as viruses and antibodies, the load remains basically unchanged. Can be conjugated in either the aqueous or organic phase, and the Epoxy agarose beads are fully swelled into ready-to-use gel beads.

2. Product characteristics

Product Name	Magarose-Epoxy，Magarose-A-Epoxy
Item number	PMAG014，PMAG 015
Magnetic bead concentration	25% (v/v)  in 50% DMSO
ligand density	60~70 μmol Epoxy/ml magnetic beads
Medium	6% cross-linked magnetic agarose
Particle size	20-45μm
Ligand binding	> 20 mg BSA/ml magnetic 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. Product features

w Mild coupling conditions (pH 8-10, temperature 4-45°C, time 1-16 hours, can be conjugated in aqueous or organic phases), high coupling efficiency (ligand density 60-70 μmol/mL).

w Magnetic beads do not need to be processed, directly calculate the appropriate amount for coupling, shorten the time.

w Long storage time, -20°C can be stored for 2 years, and cannot be damp.

w Rigidity and fast flow rate (300 cm/h, 100 kPa).

w It can be conjugated to polysaccharides containing amino, sulfhydryl and hydroxyl groups, proteins, nucleic acids, antibodies and other ligands, and has a wide range of applications.

w The affinity packing prepared after conjugation has a long life, fast flow rate and high specificity.

2. Protein coupling operation steps

Take the Epoxy agarose magnetic beads conjugated to bovine serum albumin (BSA) as an example:

1) Take 20 ml of activated agarose magnetic beads, magnetically separate, and wash three times with 50 ml of deionized water. Magnetic separation to remove supernatant.

2) Dissolve 100 mg of BSA in 10 mL of 0.25M sodium carbonate buffer at pH 9.0.

3) Add the BSA solution to the magnetic beads and mix, shake on a shaker, and couple at 25°C for 6 hours.

4) Then add 50 mg glycine and react for another 6 hours to block the remaining groups.

5) The magnetic beads conjugated to BSA were washed 3-5 times with 1 M NaCl solution, the remaining liquid after conjugation and the washed solution were collected to determine the total amount of BSA, and then the magnetic beads were washed 3 times with pure water, and finally the magnetic beads were stored in the corresponding protein preservation buffer.

6) The conjugation amount of BSA is 25 mg/ml. Calculation method: The adsorption load of BSA = (G0—G1)/V.

G0: Total BSA usage, unit: mg;

G1: Amount of unconjugated BSA after conjugation reaction, unit: mg;

V: Volume of packing material, unit: mL;

Note: This conjugation method can be used to conjugate monoclonal antibodies and other proteins, and can obtain good conjugation effects. If the conjugated substance is a small molecule such as amino acids or other small molecule ligands with -NH 2, -SH, -OH and other groups, the ligand can be directly added to 0.05M NaOH to form a solution of 10-20mg, and other conditions remain unchanged. The temperature of the coupling can also be increased, so that the coupling density can be higher.

3. Precautions

1) The activated magnetic beads are used now to avoid the reduction of coupling activity caused by too long time. It can be sealed and stored at -20°C, so it can be stored for 2 years. If the protein to be conjugated is unstable, it can be shaken at 4°C for 12-16 h on a shaker to maintain ligand activity. If you are not sure that you can match the corresponding concentration of ligand solution 0.5-1mL, choose different pH and temperature respectively and then do not add filler to simulate the coupling conditions, and observe whether the solution has precipitation in time, if there is a precipitation coupling effect is not good, at this time you can slightly reduce the pH or temperature, and at the same time you can add about 5% glycerol or PEG protective protein to avoid precipitation, in short, it is best to understand its solubility and stability for any ligand to avoid losses caused by experimental failure.

2) Clean the magnetic beads as soon as possible after conjugating the ligand to avoid inactivation of the ligand under high pH conditions.

3) The more stable ligand can be directly dissolved in NaOH solution or sodium carbonate solution for coupling, and the temperature can be appropriately increased, and the coupling time can be shortened to 1-4 hours.

4) The optimal pH of the conjugated protein can be about 8.0-10, at room temperature or 4°C, the increase of temperature and pH can increase the adsorption capacity of the ligand, according to the stability of the protein to choose the appropriate pH and temperature, the concentration of the conjugated protein solution is 5-20 mg/ml. The volume of the magnetic beads and the ligand solution is 1:1.5 or 1:2.

5) Conjugated buffers include carbonate, borate, and phosphate buffers, but substances containing amino groups such as Tris and glycine should never be used.

6) If the conjugated substance is a small molecule compound, such as amino acids or other small molecule ligands with -NH 2, -SH, -OH and other groups, the ligand can be directly added to 0.05 M NaOH and configured into a solution of 10-20 mg/mL, and other conditions remain unchanged. The temperature of the coupling can also be increased, so that the coupling density can be higher.

7) In the coupling, the magnetic beads should be mixed evenly and completely suspended, avoiding violent stirring to reduce the flow rate with small particles in the packing.

8) This reagent can only be used for in vitro experiments, not for clinical, therapeutic and in animal experiments, and is not responsible for the consequences arising therefrom.

More on http://www.magarose.com

Client Publications:

1. Jiang S, Lin Y, Zheng S. Development of the IMP biosensor for rapid and stable analysis of IMP concentrations in fermentation broth[J]. Biotechnology Journal, 2024, 19(6): 2400040. (agarose magnetic bead-coupled protein).