I am seeking a reliable protocol for conjugating polyclonal antibodies to magnetic beads for the purpose of isolating proteins from serum. Given the wide variety of bead types available, which ones would you recommend for optimal specificity and ease of use?
Additionally, what is the most user-friendly protocol for this process? Any recommendations or references would be greatly appreciated!
Marine Ma ,
If you are looking for highly specific and easy-to-use magnetic beads for protein isolation from serum, MCE (MedChemExpress) NHS Magnetic Beads may be a suitable choice.
MCE NHS Magnetic Beads (200 nm, 10 mg/mL) are modified with N-hydroxysuccinimide (NHS) groups on their surface, which can react with primary amine groups in proteins or other biomolecules to form stable amide bonds. These beads are widely used in affinity purification of antigens, antibodies and other biomolecules.
The following is a detailed experimental operation procedure for your reference:
1. Protein preparation
Dissolve the target protein in Coupling Buffer, adjust the concentration to 0.5–2.0 mg/mL, and store at 4°C.
Note: Buffers containing primary amines will inhibit the coupling reaction between proteins and magnetic beads and should be removed by dialysis or desalting.
2. Magnetic Bead Pretreatment
Completely mix the magnetic beads by repeated inversion, gentle vortexing, or rotating the platform. Take 500 μL to a new 1.5 mL centrifuge tube, separate using a magnetic stand, and discard the supernatant. Add 1 mL of Washing Buffer, vortex for 15 seconds, separate again and discard the supernatant. Repeat this step twice.
3. Protein Coupling
Add 500 μL of protein solution to the tube, vortex for 30 seconds, place in a rotator, and incubate at room temperature for 1–2 hours.
Vortex for 15 seconds every 5 minutes for the first 30 minutes of incubation; then vortex for 15 seconds every 15 minutes until the incubation is complete. If necessary, incubate overnight at 4°C.
4. Blocking
Collect the beads using a magnetic stand and retain the flow-through. Add 1 mL of Blocking Buffer, vortex for 30 seconds, and incubate at room temperature for 2 hours with rotation.
5. Storage
Separate the beads with a magnetic stand for 2 minutes and discard the supernatant. Add 1 mL of ultrapure water, repeat the magnetic separation and discard the supernatant. Repeat the above steps three times. Finally, add 500 μL of storage buffer, mix thoroughly, and store at 4°C for subsequent use.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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