Bright fluorescence but Western blot shows very low expression (Transfection)

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Accepted Answer

GFP fluorescence is obvious under microscope, but the corresponding band on Western blot is faint or near-invisible. Common causes: 1) Plasmid expression level is intrinsically low (weak promoter) 2) Construct error (frame shift, premature stop, wrong insert) 3) Insufficient expression time before harvest 4) Target protein has fast turnover / is rapidly degraded 5) Western antibody sensitivity is too low Solutions: 1) Swap to a stronger promoter (CMV is standard; CAG / EF1α for resistant cells) 2) Sanger-sequence the construct to confirm reading frame and tag orientation 3) Extend expression window (24 – 48 h post-transfection) 4) Add a proteasome inhibitor (MG-132) briefly before harvest if degradation is suspected 5) Switch to a more sensitive antibody or detection method (chemiluminescence with HRP-amplified)