Protein extraction biological reagent AB always forms precipitate, is it because the concentration is incorrect?

A total protein concentration of 2 mg/mL in the sample is not particularly high, but if the 1:50 antibody has high affinity, it may still bind excessive protein and form complexes. You can try reducing the antibody amount to around 1:100, or calculate it based on the antibody/total protein ratio; meanwhile, change the overnight rotation at 4°C to 2–4 hours to shorten nonspecific binding time. After binding, wash thoroughly with buffer and observe whether the precipitate decreases while the recovery rate remains acceptable.

Even if the supernatant of 293T lysis appears clear, it may still contain DNA, lipoproteins, and cell debris. It is recommended to centrifuge at high speed after cracking, such as at least 15000g for 15 minutes, and then take the supernatant and pass it through a 0.45 μ m or 0.22 μ m filter membrane. If the liquid is viscous, a small amount of Benzinase nuclease can be added and treated on ice or at room temperature for 10-20 minutes to degrade the nucleic acid, followed by affinity purification. The white flocculent precipitate often decreases.

You can first run a reagent-only control: dilute A and B at 1:50 in the same buffer without adding sample, rotate overnight at 4°C, then centrifuge and check whether white flocculent precipitate appears. If it also appears, A and B themselves may aggregate under low temperature, salt concentration, or pH conditions, or the stabilizer may precipitate out. You can first centrifuge or pass through a 0.22 μm filter and try again, but filtration may cause loss of activity, so it is recommended to verify with a small amount before deciding.

The appearance of white flocs does not necessarily mean that the experiment has failed. First, centrifuge and collect the supernatant for further purification. Then, precipitate and retain the sample for SDS-PAGE or Western blotting to determine if the bands are target proteins, impurities, or antibody light/heavy chains. If the precipitate is mainly composed of miscellaneous proteins, it may be non-specific binding; But it is necessary to confirm whether the target protein in the supernatant is significantly reduced. If necessary, add 0.05-0.1% non-ionic detergent or increase the NaCl concentration to 150-300mM in the washing solution to reduce non-specific aggregation.