I'm working with MCE Protein A/G magnetic beads (HY-K0202) where both antibody and antigen are currently bound. My goal is to recover only the antigen fraction while keeping the antibody coupled to the beads (to avoid H&L chain interference). Has anyone successfully achieved this?
Hello, Marine Ma .
If you want to elute the protein without antibodies, it is recommended to use the acid elution method and use a crosslinker in the step of incubating the antibody with the magnetic beads, and then incubate with the antigen.
Specific steps
- Add an appropriate amount of acid elution buffer (such as 0.1 M glycine-HCl, pH 2.5-3.0; or 0.1 M citric acid, pH 3.0), incubate at room temperature for 5-10 minutes, and shake gently.
- Immediately after magnetic separation, transfer the supernatant to a new tube containing neutralization buffer (such as 1 M Tris-HCl, pH 8.0) and neutralize to neutral pH to avoid antigen denaturation.
- Repeat elution 1-2 times and combine the eluates.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
You need to covalently cross-link the antibody to the A/G beads. Below is a protocol we use with Sepharose-4B-Protein a/G.
1) Incubate the protein A gel (0.5mL) with the antiserum you want the antibody from or the purified antibody for 30 minutes at room temperature. Wash with PBS (without sodium azide) 5x.
2) Re-suspend the gel in 2mL of 0.1M sodium tetraborate buffer, pH 8.0. Leave for 10 minutes.
3) centrifuge the gel then add 2mL of freshly made cross-linker dimethylsuberimidate at 5mg/mL in the borate buffer. Mix for 1 hour at room temperature.
4) Wash 2x with PBS containing 0.5M NaCl. Store gel in PBS at 40C.
5) Acid wash of the gel may lead to loss of about 10% of the bound antibody
We have developed a method to crosslink antibodies to protein A or G without reducing their activity: https://doi.org/10.3390/mps2020035
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