Jan 26, 2026
Magnet-assisted immunoprecipitation (MAIP) of recombinant protein
- Marvin De los Santos1
- 1ChordexBio
Protocol Citation: Marvin De los Santos 2026. Magnet-assisted immunoprecipitation (MAIP) of recombinant protein. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld1w9xl5b/v1
Manuscript citation:
De Los Santos, M. I., & Bernal, S. D. (2024). U.S. Patent No. 11,912,751. Washington, DC: U.S. Patent and Trademark Office.
De Los Santos, Marvin I. (2019). Construction and Characterization of a Novel Bifunctional TGF-B1/PD-L1 Fusion Protein (M.S. in Miolecular Biology and Biotechnology Thesis). University of the Philippines Diliman. College of Science. National Institute of Molecular Biology and Biotechnology. UP Diliman CS Library. https://cslib.science.upd.edu.ph/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 25, 2026
Last Modified: January 26, 2026
Protocol Integer ID: 240859
Keywords: Immunoprecipitation, magnet-based protein extraction, recombinant fusion protein, TGF-β1/PD-L1 fusion protein, HEK 293 cell expression, transient protein expression, magnet-assisted immunoprecipitation, selective capture of the fusion protein, l1 fusion protein from mammalian cell lysate, l1 fusion protein, fusion protein, detailed steps for antibody coupling, assisted immunoprecipitation, antibody coupling, magnetic isolation, recovery of intact protein, protein extraction, recombinant protein, recombinant protein this protocol, intact protein, mammalian cell lysate
Abstract
This protocol describes a magnet-assisted immunoprecipitation (MAIP) workflow for the isolation of a TGF-β1/PD-L1 fusion protein from mammalian cell lysates under native conditions. Dynabeads M-280 Tosylactivated are covalently coupled with either anti-TGF-β1 or anti-PD-L1 antibodies to enable selective capture of the fusion protein following transient expression in HEK 293 cells. The method includes detailed steps for antibody coupling, protein extraction, immune-magnetic isolation, acidic elution, and neutralization, allowing recovery of intact protein suitable for downstream biochemical analyses such as SDS-PAGE and immunoblotting.
Troubleshooting
Magnetic beads preparation
2d 17h 9m 40s
Prepare the magnet-coated beads. For this protocol, the Dynabeads M-280 Tosylactivated beads are used
2d
Resuspend the Dynabeads in the vial by vortexing for 00:00:30
30s
Transfer 300 µL of the beads to a new, sterile 1.5 mL tube
Add 1 mL of Buffer B to the tube and vortex for 00:00:30
| A | B | C | |
| Reagent | Molecular weight (g/mol) | Amount needed | |
| NaH2PO4 × H2O | 137.99 | 2.62 g | |
| Na2 HPO4 × 2 H2O | 177.99 | 14.42 g | |
| NaOH or HCl | – | pH 7.4 | |
| Distilled water (H2O) | 18.015 | Fill to 1 L |
Buffer B composition
30s
Place the tube in a magnet for 00:01:00 and discard the supernatant. Repeat once
1m
Remove the tube from the magnet and resuspend the washed beads in 300 µL of Buffer B. Divide the washed beads into two tubes, containing 150 µL each
Couple the antibodies covalently with Dynabeads M-280 Tosylactivated using the recommended protocol from the manufacturer. For this protocol, the anti-TGF-β1 and anti-PD-L1 antibodies will be used. However, different antibody reagents can be subjected for coupling
Place the tubes in a magnet for 00:01:00 and discard the supernatant
1m
For every 150 µL of Dynabeads as the initial volume, resuspend in 100 µg/150 µL of antibodies (anti-TGF-β1 or anti-PD-L1) diluted using Buffer B. Mix by vortexing for 00:00:30
30s
Add 100 µL Buffer C and mix by vortexing for 00:00:30
| A | B | |
| Reagent | Amount needed | |
| [(NH4)2]SO4 | 39.64 g | |
| Buffer B | 100 mL | |
| NaOH or HCl | pH 7.4 |
Buffer C composition
Incubate on a shaker at 37 °C for 16:00:00
16h
Place the tube on a magnet for 00:02:00 and remove the supernatant
2m
Remove the tube from the magnet and add 1 mL Buffer D; incubate at 37 °C for 01:00:00 on a shaker.
| A | B | |
| Reagent | Amount needed | |
| NaCl | 0.88 g | |
| BSA (Bovine Serum Albumin) | 0.5% (w/v) | |
| Buffer B | 80 mL | |
| NaOH or HCl | pH 7.4 |
Buffer D composition
1h
Place the tube on a magnet for 00:02:00 and remove the supernatant.
2m
Remove the tube from the magnet and add 1 mL Buffer E; vortex for 00:00:10
10s
Place the tube on a magnet for 00:02:00 and remove the supernatant.
2m
Repeat steps and once.
For every 150 µL of Dynabeads, resuspend and dilute the beads in 200 µL of Buffer E.
| A | B | |
| Reagent | Amount needed | |
| NaCl | 0.88 g | |
| BSA (Bovine Serum Albumin) | 0.1% (w/v) | |
| Buffer B | 80 mL | |
| NaOH or HCl | pH 7.4 |
Buffer E composition
Protein extraction
2d 0h 11m
Following transfection, harvest HEK 293 cells after 48:00:00 of incubation.
2d
Decant the culture medium and wash the cells twice with sterile PBS.
Submerge the cells in PBS and incubate at 25 °C for 00:01:00
1m
Tap the sides of the plate gently to detach the cells and transfer the supernatant.
Collect the cells by centrifugation at500 x g, 25°C, 00:05:00
5m
For every 1 × 10⁶ cells, resuspend using 500 µL of native protein extraction buffer.
| A | B | |
| Reagent | Amount needed | |
| Tris | 50 mM | |
| EDTA | 1 mM | |
| SDS | 0.001% (w/v) | |
| Tween-20 | 0.01% (w/v) | |
| NaCl | 50 mM |
Composition of the native protein extraction buffer, pH 7.4
Lyse the cells by passing the solution through a 23-G needle syringe 15 times. This should be done gently and on ice.
Clarify the lysate by centrifugation at 500 x g, 25°C, 00:05:00 . Transfer the supernatant to a new sterile tube, and store the pellet at -80 °C for future protein recovery.
5m
Quantify the protein content of the extract. Any protein quantification method available in the laboratory may be used.
Immuno-magnetic isolation
37m 10s
Add 10 µg of sample containing the recombinant protein to 50 µL of the antibody-coupled beads. For this protocol, a bifunctional fusion protein that contains the TGF-B1 and PD-L1 domains are used. Prepare two separate reactions for each antibody-coupled Dynabeads (anti-TGF-β1 or anti-PD-L1)
Incubate with tilting and rotation for 00:30:00
30m
Place the tube on a magnet for 00:02:00 and pipette off the supernatant
2m
Remove the tube from the magnet and add 1 mL PBS; vortex for 00:00:10
10s
Place the tube on the magnet for 00:02:00 and remove the supernatant
2m
Repeat steps and twice
Add 50 µL of 100 mM glycine-HCl (3.0 ) and incubate for 00:01:00
1m
Place the tube on the magnet for 00:02:00 and collect the supernatant
2m
Neutralize the collected supernatant by adding 5 µL of 1 M Tris-HCl (8.8 ). At this point, the beads may also be washed with 50 µL of 1 M Tris-HCl for reuse in future isolations.
Quantify the concentration of the isolated protein and analyze the purity using SDS-PAGE or other methods