If I inject mice with wild-type cancer cells that naturally express multiple genes, we know their immune systems will produce antibodies against nearly all the genes present in those cancer cells. Now, I’ve isolated the B-cells from these mice and cultured them. How can I measure the antibody concentration in the culture supernatant without interference from other cellular components?
I’ve been culturing these cells in low-IgG FBS - is there anything else I should consider? Also, I’m planning to filter the supernatant using 100K spin columns since I assume the antibodies produced by the murine B-cells are around 150 kDa, then take the concentration using a NanoDrop A280.
Does this approach sound correct?
I’d really appreciate any advice or suggestions!
Hi, Erica Weber.
It is not recommended to perform ELISA testing through specific targets because the immunogen is a "wild-type cancer cell that naturally expresses multiple genes".
You can try to purify the supernatant with Protein A/G Magnetic Beads, and then perform SDS-PAGE purity identification + A280 detection concentration.
MCE provides reliable Protein A/G Magnetic Beads (HY-K0202), which has the following advantages:
1. Only a small amount of magnetic beads is required for immunoprecipitation.
2. Convenient and time-saving.
3. Low non-specific binding rate.
4. Less sample loss.
5. Protein binding capacity is as high as 0.7 mg/mL.
6. Stable.
The specific operation process can be found on the MCE official website (https://www.medchemexpress.com/inhibitor-kit/protein-a/g-magnetic-beads.html).
The process directory is as follows:
1. Antigen sample preparation.
2. Magnetic bead pretreatment.
3. Antibody binding to magnetic beads.
4. Antigen binding to antibody-magnetic bead complex.
5. Antigen elution.
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