Two researchers conducted a Western blot analysis to determine the expression level of a specific protein in cultured cells. They used the same membrane, the same primary and secondary antibodies, and the same imaging system. Researcher 'X' reported a protein band intensity corresponding to a relative expression level of 200 units, while researcher 'Y' reported 800 units. Which one of the following could most likely explain the discrepancy in researcher 'Y's result?

1

The membrane was not washed properly after the primary antibody incubation.

2

The membrane was exposed to the imaging system for a longer duration than necessary.

3

The standard curve used for quantification was incorrectly plotted.

4

The loading control bands were not properly visualized.

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