Can domestic alternatives be used for WB primary antibody diluent? How much will the titer drop with repeated use?

Repeated recycling will cause a drop in titer, but how much it drops depends on the antibody type, the buffer capacity of the diluent, and the storage conditions; it is not a fixed ratio. For high-abundance protein primary antibodies, within 3-5 rounds of recycling, the decrease in the internal control band is usually not easy to see with the naked eye; low-abundance or phosphorylated antibodies may show noticeably weaker signal after only 1-2 uses. It is recommended to label each recycling with: antibody name, number of recycles, storage date, and which protein it is used for. To make a judgment, run parallel gels or cut the membrane, incubate the freshly diluted primary antibody and the recycled solution at the same time on different lanes of the same membrane, and fix the color development time to observe grayscale. If the band from the recycled solution is noticeably weaker than that from the freshly diluted solution, or if the target band becomes lighter while the internal control does not change, it indicates that the titer has decreased. Adding sodium azide can only inhibit bacteria; it cannot restore the titer, and mixing old and new is not recommended.

For storage and aliquoting, I recommend primarily 4°C. With 20 samples per week and frequent use, repeated freeze-thaw cycles can easily cause the protein stabilizer or surfactant to separate, leading to batch-to-batch variation. Aliquot into 10-15 mL tubes based on one week's usage, store at 4°C, and use up in the short term; for long-term non-use, then store at -20°C or -80°C. Regarding preservation, adding sodium azide to the primary antibody diluent at a final concentration of 0.02%-0.05% can inhibit bacteria, but note that sodium azide inhibits HRP. If the membrane is not washed sufficiently and residue carries over to the secondary antibody color development, it will have an impact. Therefore, for primary antibody diluent containing sodium azide, the TBST washing time after incubation must be sufficient, and do not add it to the secondary antibody diluent. If not using sodium azide, do not store at 4°C for too long. If turbidity or odor is found upon opening, discontinue use.

Domestic alternatives are not unusable, but parallel validation is recommended first. Find 1-2 domestic brands with good reputations and small packaging, choose a formulation containing BSA or gelatin, Tween-20, phosphate buffer, and preservatives, and use it simultaneously with your current imported diluent to dilute the same primary antibody, with the same batch of lysis buffer, the same concentration, and the same incubation time, and fix the exposure time during ECL development. Focus on the gray values of the internal control and target bands as well as the background. Low-abundance proteins or phosphorylated antibodies are more sensitive to the diluent, and such indicators should be validated separately. If the trends of the internal control and target are consistent and the background does not increase, you can switch to the domestic product first, run non-critical samples for two weeks, and then switch completely. Do not switch everything at once, to avoid batch or formulation differences affecting the entire dataset.