1 Preparation of protein solution
Take an appropriate amount of the protein to be coupled and dissolve it in Coupling Buffer to prepare a protein solution with a concentration of 0.1-3.0 mg/mL. The protein that has been stored in the buffer needs to be completely removed by dialysis or desalting to remove the primary amine-containing substances in the original buffer, and then use Coupling Buffer to prepare a protein solution with a concentration of 0.1-3.0 mg/mL. The protein solution is stored at 4oC for use.
a. In the process of protein coupling, you must first determine the appropriate Coupling Buffer.
b Protein solution cannot contain components with primary amino groups, such as Tris, glycine, gelatin, BSA, etc.
2 Magnetic Beads Pretreatment
2.1 Take 500 μL magnetic beads into a 1.5 mL EP tube.
Note: The magnetic beads should be inverted repeatedly before sampling, use a vortex oscillator or a vertical mixer to make the mixture evenly
2.2 Place the EP tube in the magnetic separation rack, enrich the magnetic beads and remove the supernatant.
2.3 Add 1 mL of 2~8oC pre-cooled Wash Buffer A to a 1.5 mL EP tube and vortex for 15 s to mix the magnetic beads evenly.
2.4 Put the EP tube in the magnetic separation rack, enrich the magnetic beads, and remove the supernatant.
3.1 Add 500 μL of protein solution to the EP tube and vortex for 30 s to mix well.
Note: Add protein solution immediately after washing the magnetic beads.
3.2 Vortex the EP tube for 15 seconds, place it on a vertical mixer, and mix at room temperature for 1-2 h. If the vertical mixing is not uniform, remove the EP tube and vortex for 15 seconds every 5 minutes 30 minutes before the reaction. Remove the EP tube and vortex for 15 seconds every 15 minutes.
Note: If necessary, react at 4oC overnight.
3.3 Use a magnetic separation to enrich the magnetic beads and store the flow-through liquid.
4.1 Add 1 mL Blocking Buffer to the EP tube, vortex for 30 seconds, place the EP tube in the magnetic separation, enrich the magnetic beads and discard the supernatant.
Note: Blocking Buffer can also use other blocking reagents such as 100 mM Tris-HCl, 150 mM NaCl, pH 8.0.
4.2 Repeat "Step 4.1" four times.
4.3 Add 1 mL Blocking Buffer to the EP tube, vortex for 30 s, and place the EP tube in a vertical mixer to react at room temperature for 2 h.
4.4 Place the EP tube in the magnetic separation, enrich the magnetic beads and discard the supernatant.
4.5 Add 1 mL of DI water to the EP tube, mix thoroughly, concentrate the magnetic beads with magnetic separation, and discard the supernatant.
5.1 Add 1 mL Storage Buffer to the EP tube, mix well, concentrate the magnetic beads with a magnetic stand, and discard the supernatant. Repeat 2 times
5.2 Add 500 μL Storage Buffer to the EP tube, mix well, and store at 4oC for later use.
Note: The final concentration of magnetic beads after protein coupling is 10 mg/mL.
Note: The operation process is described by taking 500 μL of magnetic bead sample and using 1.5 mL EP tube as an example. Users can adjust proportionally according to their own needs.
Washing Buffer A: 1 mM hydrochloric acid, keep 4oC before use
Coupling Buffer A: 100 mM 2-morpholineethanesulfonic acid (MES), pH 4.8
Coupling Buffer B: 200 mM NaHCO3, pH 8.3
Coupling Buffer C: 50 mM boric acid solution, pH 8.5
Coupling Buffer D: 100 mM phosphate buffer, 100 mM NaCl, pH 7.4
Blocking Buffer: 3 M ethanolamine, pH 9.0
Storage Buffer: 1×PBS, 0.1% proclin-300 can be added as required