Abnormal fluorescence localization (aggregates / wrong compartment) (Transfection)

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Fluorescence forms aggregates inside cells, or localizes to a compartment different from the expected native pattern. Common causes: 1) Overexpression saturates the localization machinery → mislocalization 2) Fluorescent tag is positioned in a way that disrupts targeting signals (N- vs C-terminal matters) 3) The tag itself perturbs protein folding or trafficking 4) Cell state is poor and trafficking is disrupted Solutions: 1) Lower the amount of plasmid transfected to reduce overexpression artifacts 2) Try moving the fluorescent tag from N- to C-terminal (or vice versa), or use a smaller tag (HaloTag, SNAP-tag) 3) Validate with a co-localization experiment using a known marker 4) Use cells in better state and optimize transfection conditions