Will 1% Triton X-100 interfere with the measurement of cell lysate protein concentration using BCA assay kit?

The standard curve must be matrix-matched. Do not use BSA standards prepared in PBS or pure water to quantify lysate containing 1% Triton. The correct approach is to dilute the BSA standards in exactly the same buffer system as your lysate, containing the same 1% Triton, salt concentration, and protease inhibitors. This way, even if Triton slightly inhibits or enhances color development, it is already accounted for in the standard curve. You have 20 samples at 50 μL each, so preparing a matrix-matched standard curve will not require much volume. It is best to also include a blank well containing no protein but the same lysate components, to check whether the baseline is elevated. If the blank OD is significantly high, it indicates a large background contribution that needs to be subtracted separately.

Spike recovery can be used to verify whether Triton interference is acceptable. Take a portion of the sample and add a known amount of BSA, for example, to increase the final concentration by 0.5 μg/μL, and measure the recovery. If the recovery is roughly 90%-110%, it indicates that the BCA result is still usable in this matrix; if the deviation is obvious, the Triton concentration must be reduced or the pretreatment must be changed. Another approach is to precipitate the protein with acetone or chloroform/methanol, remove the detergent-containing supernatant, and then redissolve for quantification, but the procedure is cumbersome, and with 20 samples, losses and operational errors are easily amplified. If this type of lysis buffer is encountered frequently in subsequent work, a detergent-compatible BCA or RC DC reagent can be kept on hand for comparison, but do not directly compare absolute OD values across brands.

If the tolerance level is uncertain, it is recommended to dilute with three dilutions of 1:5, 1:10, and 1:20 using the same lysis buffer, and then check if the concentration of the original solution is consistent. 1: The similarity between the 5 and 1:10 regression results indicates that the dilution has reduced Triton interference to an acceptable range; If the 1:20 signal is close to blank, it indicates insufficient detection limit and blind dilution is not recommended. Your sample size is not large, 50 μ L is sufficient for several dilution conditions and wells. Do not directly choose high fold dilution for low abundance samples, otherwise the protein concentration will fall below the detection limit. If it is found that a lot of dilution is necessary to eliminate interference, high-sensitivity BCA or compatible detergent quantitative methods should be used instead of hard testing.

First, look at the final concentration of the reaction, not the initial concentration in the lysate. Conventional 96 well BCA plates usually add 10-25 μ L of sample to 200 μ L of working solution, and 1% Triton X-100 is diluted to about 0.05% -0.1%. Many BCA formulas can tolerate this final concentration, but there may be differences among different brands and protein matrices. It is recommended to first take 2-3 representative cracking solutions for gradient dilution, such as the original solution, 1:2, 1:5, to see if the calculated concentration is linear. If the reading of the original solution is too high or not proportional after dilution, it indicates significant matrix interference and cannot be directly used. Also observe whether the working fluid becomes turbid, abnormally turns green, or precipitates after being added. If there is obvious interference, priority should be given to reducing the sample volume or dilution to solve it, rather than directly changing the method.