Can the reagent preparation ratio be halved for the 96 well BCA method, which only measures 20 samples at a time?

The system can be scaled proportionally, but the 50:1 ratio of liquid A and liquid B cannot be changed. For example, in the original system, adding 25 μ L of sample to 200 μ L of working solution can be changed to adding 12.5 μ L of sample to 100 μ L of working solution, or adding 10 μ L of sample to 100 μ L of working solution. When the sample volume is sufficient, it is recommended to add 25 μ L of sample to 100 μ L of working solution, which will make the color intensity and liquid level more stable; When the sample size is tight, 10 μ L of sample can be added to 100 μ L of working solution, but the detection limit will be increased, and the error of low concentration samples will increase. Before switching to a half volume system, compare the linearity of the standard curve with the calculated concentration of the 200 μ L and 100 μ L systems using the same batch of samples. If the deviation is acceptable, use it formally.

It is not recommended to use fewer than 6 standard curve points, including the blank. Using fewer samples saves only a small amount of working solution, but too few standard curve points will distort the fit at the low- or high-concentration end, directly affecting the IP loading amount. It is recommended to keep seven points: blank, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 mg/mL. Removing 0.025 or adding 0.75 is acceptable, but do not keep only 3-4 points. Standards can be run in single wells, but samples should be run at least in duplicate, and duplicate wells are especially important for low-concentration samples. If you want to reduce working solution waste, the saved volume should be prioritized for replicate sample wells rather than reducing standard curve points.

After reducing the volume, the errors in pipetting and the impact of evaporation will increase. It is recommended to use a calibrated 100 μ L or 200 μ L pipette. The working fluid should be pre mixed uniformly before adding, and A and B should not be added separately to each hole. When adding the fluid, press it slowly against the wall to avoid generating bubbles. Do not place samples on the outer circle of the 96 well plate. PBS or water can be added to reduce edge effects. When incubating at room temperature with a volume of 100 μ L, the evaporation ratio is higher than that of 200 μ L, and it must be covered or sealed with a plate film. Before reading the board, check if the liquid level is flat, as bubbles can significantly interfere with the OD reading. When the liquid level is low, attention should also be paid to whether the optical path of the enzyme-linked immunosorbent assay (ELISA) is sensitive to the height of the liquid level.

Before switching systems, perform a comparative validation and do not directly use it for IP quantification. Take 3-5 representative samples, prepare standard curves and samples in both 200 μL and 100 μL systems, and switch only after the calculated concentration deviation is preferably within 10%; if it exceeds this range, check the sample loading volume, incubation time, and plate reading conditions. If the signal of low-concentration samples is too weak in the 100 μL system, consider a half-area plate or high-sensitivity BCA rather than simply increasing the sample volume and disrupting the system. IP loading is sensitive to protein amount, so it is recommended to include a quality control sample of known concentration in each quantification to facilitate assessment of batch-to-batch drift.