How effective is the purification of His tagged proteins using pre installed chromatography columns from domestic biosensing brands?

When choosing a His column, pay attention to the resin type. IDA has high loading capacity but is sensitive to imidazole competition, while NTA has good resistance to reducing agents and is more suitable for samples containing DTT or β-mercaptoethanol. You can first purchase a 1 mL domestic prepacked column for a small-scale test, load it in parallel with the same batch of lysate using an imported column, and compare the breakthrough curve, eluted target protein concentration, SDS-PAGE purity, and column pressure. Note that the column volume should match a 20 mL load: a 1 mL column can be loaded repeatedly but is time-consuming, while a 5 mL column allows a single load but causes more obvious sample dilution. Different batches of domestic resins may differ in particle size, so perform a column efficiency test upon arrival.

Whether the decrease in column efficiency is significant after repeating three times depends on the cleanliness of the sample and the regeneration procedure. If the lysate is clarified, treated with protease inhibitors and nucleases, the decrease in load after three cycles is generally acceptable; If the recovery rate or peak area of the target protein decreases by more than 20% -30% compared to the first time, attention should be paid. Wash non-specific binding with high imidazole buffer solution after each use, and then perform NaOH or manufacturer recommended regeneration to avoid prolonged high-intensity soaking. It is more important to record the column pressure, recovery amount, and elution peak area each time, and observe the trend rather than just looking at a single time.

Domestic pre loaded Ni columns are commonly labeled with a loading capacity of 20-40 mg/mL. Your protein expression level is 5-10 mg/L. The total amount of target protein in 20 mL lysis buffer is about 0.1-0.2 mg. Even if there are many impurities, it is generally not overloaded. Load capacity is not the main risk point, the key is that the lysis buffer should be centrifuged at high speed and filtered through a 0.45 μ m membrane before loading to reduce blockage by nucleic acids, lipoproteins, and cell debris. We have produced His tagged recombinant proteins, and there is not much difference between domestically produced columns and imported ones at low sample sizes; But if the sample contains EDTA, high concentration reducing agents, or imidazole, the conditions should be adjusted according to the type of medium.

For flow rate and elution purity, it is recommended to follow the instructions first. For 1 mL or 5 mL columns, a common flow rate is 1-2 mL/min. Loading 20 mL of sample onto a 5 mL column at one time is fine, but the loading time is relatively long. You can perform cyclic loading at 4 degreesC or slow down the flow rate to extend the binding time. The key to elution purity lies in the wash conditions: 20-40 mM imidazole for washing impurities, and 250-500 mM imidazole gradient elution. The elution peak of domestic columns may be slightly broader than that of imported ones. Whether this is acceptable depends on the purity of the target band and the recovery rate in SDS-PAGE. For comparison, use the same batch of lysate for parallel loading to avoid interference from lysis differences in the judgment.