The endotoxin level of His-tagged recombinant protein is relatively high. How can it be removed in the laboratory?

Don't rush to attribute all negative control activation to protein endotoxin. In macrophage experiments, serum, culture medium, pipette tips, and culture dishes can all carry LPS. It is recommended to first perform an endotoxin contamination screening: reconstitute the same batch of TNF-α with endotoxin-free water, and set up groups with added polymyxin B, TLR4 blocking antibody, or inhibitor. If the negative control activation is reversed after adding polymyxin B, it more strongly supports LPS-type contamination; if there is no change, it may be the protein itself or operational factors. With a budget of 1500 per vial, it is advisable to spend a small portion of that money on endotoxin-free consumables and LAL limulus reagent, first quantify the endotoxin level, and then decide whether to return/exchange or switch to a low-endotoxin batch. Otherwise, after spending ages running columns yourself, the problem might be the pipette tips.

For macrophages, especially THP-1 or BMDM, TNF-α at 1 EU/μg will almost certainly be interfered with by LPS contamination, because the TNF you use to stimulate and LPS have a synergistic effect. With a budget of around 1500, prioritize batches labeled as less than 0.1 EU/μg or even lower, and ask the manufacturer for measured endotoxin data and the LAL method. If the lab removes endotoxin from His-tagged proteins itself, Triton X-114 extraction will cause protein loss and introduce detergent, making the impact on subsequent cell status difficult to control; affinity columns or polymyxin B endotoxin removal columns are feasible but need to be combined with endotoxin-free operations, and recovery is often only 50% to 80%. The amount used per vial is small, so risking experimental failure to save a few hundred yuan is not worthwhile.

If you truly cannot purchase a low-endotoxin batch, or want to salvage existing stock, you can use polymyxin B agarose columns or endotoxin removal resin. The key is to make the column, buffers, and collection tubes all endotoxin-free, such as by baking at 180°C or purchasing pyrogen-free consumables. Before loading, measure the endotoxin level in the protein solution using LAL reagent. Typically, endotoxin needs to be reduced from 1 EU/μg to less than 0.1 EU/μg, which may require passing through the column twice. TNF-α is a cytokine, and its activity will decrease after column passage. It is recommended to perform protein quantification and a macrophage activity control in parallel. Endotoxin removal columns have requirements for buffer pH and salt concentration; avoid pure water or low salt, which can cause protein precipitation. If the concentration becomes lower after recovery, do not blindly increase the volume for stimulation; calculate according to the actual activity units.