I'd like to ask everyone which brand of prepacked columns is more durable for His-tagged protein purification?

A decline in capacity after seven or eight cycles does not necessarily mean the resin is failing; it may be due to metal ion leaching or column clogging from precipitated host cell proteins. After a new column is put into use, it is recommended to establish a fixed regeneration protocol: first strip nickel with a buffer containing 50 mM EDTA, then clean deposited proteins with 0.5 M NaOH or 6 M guanidine hydrochloride, and finally re-charge with Ni. Samples containing DTT should not be left in contact with the column for extended periods; elute and regenerate as soon as possible after loading. 20 mM imidazole can continue to be included in the equilibration/wash buffer to suppress nonspecific binding, but samples should be centrifuged or filtered through a 0.45 μm membrane before loading to avoid clogging. This typically extends the lifespan of columns of the same model considerably.

If you want durability within 3,000 RMB, consider buying bulk resin and packing your own column, such as Cytiva Ni Sepharose 6 Fast Flow or the HP series. Look for authorized domestic reagent distributors, many of which can ship from stock within a week. Packing your own gravity column or small XK column costs less than a prepacked column, and the bed height and loading capacity are easier to control. If you are concerned about DTT interference, choose a reducing-agent-resistant model or switch to a milder sample buffer system. For the first purchase, buy a small amount of resin to test dynamic binding capacity and column pressure, then scale up with the same lot. For suppliers, you can compare authorized channels from Cytiva, Bio-Rad, and Thermo Fisher, and request COA as well as resin particle size and nickel density parameters.

The sample contains 5 mM DTT and 20 mM imidazole. With regular Ni-NTA, nickel leaching accelerates with prolonged use. It is recommended to prioritize reducing agent-resistant Cytiva HisTrap Excel series, or TALON cobalt columns (Takara/Clontech). Cobalt columns generally tolerate DTT somewhat better than nickel columns. Your loading volume is around 50 mL. If the lysate is relatively clear, you can first test binding capacity and pressure with a 1 mL small column; within budget, you can also use a 5 mL Excel prepacked column. When purchasing, confirm lead time with Cytiva authorized distributors or regional stockists, and request documentation on the tolerance concentrations of DTT and β-mercaptoethanol for that specific model.