1. Overview

Magarose-ProA (ProG, ProA/G, ProL) antibody purification beads are composite particles formed by covalently conjugating NHS-activated magnetic agarose microspheres to Protein A (or Protein G). Compared with similar products in the current international immunomagnetic bead market, this product has higher antibody binding capacity and lower protein non-specific adsorption rate, and the elution conditions are more uniform, and antibodies with a purity greater than 90% can be isolated from serum samples in one step. This product is a micron-level magnetic microsphere, which can complete the antibody adsorption process within 15 minutes and the antibody purification process within 30 minutes with skillful operation. This product is reusable and is suitable for antibody purification in plasma, ascites, tissue culture supernatant and other samples, as well as for antibody fixation and other related studies. Users can select the bead class based on the species source and isotype of the target antibody, see http://www.purimagbead.com/html/4309815423.html for comparison of the affinity of Magarose-ProA (roG, ProA/G, ProL) magnetic beads with different antibodies.

2. Product characteristics

Product Name	Magarose-ProA （ProG、ProA/G、ProL）
Item number	PMAG008
Magnetic bead concentration	25% (v/v) in PBST (containing 0.05% NaN3).
ligand density	/
Medium	6% cross-linked magnetic agarose
Particle size	20-45μm
Ligand binding	~40 mg Human IgG/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. Buffer

Binding/Washing buffer PBST（pH 7.2~7.4）: 137 mM NaCl，2.7 mM KCl, 10 mM Na₂HPO₄, 2.0 mM KH₂PO₄, 0.1% Tween-20;

Elution buffer: 100 mM Glycine, 0.1% Tween-20, pH 2.5;

Neutrilization buffer: 1.0 M Tris-HCl, pH 9.0;

2. Procedure (taking purified human serum IgG as an example).

1) Sample processing: Take 100 μL of human serum into a 1.5 mL EP tube, then add 900 μL of Binding/Washing buffer and mix well.

2) Magnetic bead pretreatment: The antibody purified magnetic beads were swirled and shaken for 30 s to fully resuspend the beads. Place 100 μL of 10% (v/v) magnetic bead suspension in a new 1.5 mL EP tube. The magnetic bead suspension was magnetically separated, the supernatant was discarded, and washed twice with a 1 mL Binding/Washing buffer, and the magnetic beads in the tube could be directly used for antibody separation.

Note: The amount of magnetic beads in this step can be adjusted according to the maximum binding amount of the target antibody by the magnetic beads, when the concentration of the target antibody is greater than 150 μg/mL, the user can take 1.2~1.5 times the magnetic bead dosage (calculation method: such as 1.5 * target antibody content in the sample/maximum binding amount of magnetic beads), if the target antibody concentration is too low, such as less than 70 μg/mL, the customer can increase the magnetic bead dosage to improve the antibody recovery, such as increasing to 3 times the magnetic bead dosage.

3) Antibody adsorption: Add the sample solution treated in step 1 to the magnetic bead tube pretreated in step 2, swirl evenly, place it in a flip mixer or gently flip the EP tube at room temperature (about 25°C) to promote full contact with the magnetic beads and adsorption, and after about 15 minutes of flipping, magnetic separation is carried out, and the supernatant is discarded.

4) Magnetic bead washing: Add 1 mL Binding/Washing buffer to the EP tube, shake and resuspend the magnetic beads for magnetic separation, and discard the supernatant. This operation is repeated 3 times.

5) Antibody elution: Add 0.5~1.0 mL Elution buffer to the EP tube with magnetic bead washing, quickly resuspend it with a pipette or vortex shaking, then place it in a flip mixer at room temperature (about 25°C) or gently turn the EP tube by hand, turn it over for 10 minutes and then perform magnetic separation, and collect the supernatant into a new EP tube.

Note: For the amount of Elution buffer in this step, it is recommended that customers control the concentration of antibodies in the final elution at 0.6~1.2 mg/mL, at which time more than 95% of the antibodies in the first elution condition will be eluted; If the elution buffer is used too little, some of the antibody will remain on the beads during the first elution, resulting in reduced antibody recovery.

6) Antibody neutralization: Add a certain amount of Neutrilization buffer to the antibody eluent in step 5, generally 1/10 of the antibody elution volume, and finally keep the pH value of the eluted antibody in a neutral environment, so as to help maintain the biological activity of the antibody and avoid antibody inactivation.

7) Post-treatment of magnetic beads: After use, the magnetic beads are washed twice with Elution buffer, magnetically separated, and the supernatant is discarded; Then wash 3 times with a binding/washing buffer, magnetically separate, discard the supernatant, add 100 μL of storage buffer to resuspend the beads, and store at 2~8°C.

3. Magnetic bead regeneration

1) After multiple uses, impurities such as precipitated protein, strong hydrophobic protein, lipoprotein and other impurities will be non-specifically adsorbed on the magnetic beads, in order to ensure the efficiency of the use of magnetic beads, it is recommended to continue to use them 5 times before regeneration of magnetic beads.

2) Add 2 mL of 1% (v/v) Triron X-100 magnetic bead regeneration buffer per 1 mL of 25% (v/v) magnetic beads, shake evenly, place in a flip mixer at room temperature or gently flip and mix by hand, and then magnetically separate after 10 minutes, and discard the supernatant.

3) Immediately add 1 mL of Binding/Washing buffer for resuspension, then magnetically separate, discard the supernatant, and repeat the operation 3 times.

4) Add 1 mL of Storage buffer to resuspend the magnetic beads and store at 2~8°C.

More on http://www.magarose.com