Is it a serum batch issue that the WB background has increased after changing the serum brand?

Cell proliferation looks acceptable, so I do not recommend immediately switching back or treating the serum. First let the cells adapt for 3-5 passages, and set up three parallel groups: old serum, new serum, and old:new 1:1. Collect samples every 2 passages and run them on the same WB. If the background increases with the proportion of new serum, it suggests a relationship with batch components; if the 1:1 group is also high, go back and check the blocking solution, membrane washing solution, and secondary antibody diluent. I have encountered increased background after changing serum due to changes in cell membrane lipid composition, not an antibody problem. You can also add 10% of the new serum to the blocking solution for one absorption step and see whether the background decreases. Endotoxin and hemoglobin can be measured, but do not focus only on these two indicators.

From an operational perspective, first troubleshoot the samples and buffers. If HepG2/RAW264.7 passage is normal but WB background is high, it may be due to increased endogenous biotin, lipoprotein, or IgG binding in the cells after culture with the new serum. It is recommended to reduce the loading amount, change the blocking solution to 5% BSA overnight at 4°C, increase Tween-20 in TBST to 0.1%-0.2%, and increase the number of membrane washes to 5×5 min. Then perform serum cross-validation: collect samples simultaneously from cells cultured with the old serum and cells cultured with the new serum, and compare the background; if the background decreases after switching back to the old serum for 2 passages, this indicates that the serum factor is significant. Domestic serum can be subjected to 56°C 30 min inactivation, 0.22 μm filtration, or 10 kDa dialysis, and then another group can be cultured to observe the differences. Measure endotoxin/hemoglobin as a supplement, but this is not a sufficient explanation for the high background.

Background elevation is associated with the serum lot, but do not draw conclusions directly. Common causes include residual IgG, complement, or lipids in the new serum, leading to blocking/secondary antibody cross-reactivity, or altered protein expression profiles after cell stress. Three validation steps are recommended: 1) Add the new serum and old serum separately to blocking solution or secondary antibody diluent, run a gel/dot blot directly to see whether background is generated; 2) Treat cells with 0.5% BSA or serum-free medium for 24 h before harvesting samples to rule out residual serum interference; 3) Measure endotoxin and hemoglobin in that serum lot, and if conditions permit, perform 10 kDa dialysis or 56°C inactivation, then culture in parallel for 2-3 passages before harvesting for WB. If the background reproduces with addition of the new serum, it can basically be attributed to that lot.

It is likely that complement or antibody components in the serum are affecting the background; try switching back to the old lot.

Check the blocking solution and antibody concentrations first; don't rush to blame the serum.