Does GFP interferes with absorbance in MTT assay or luminescence in Caspase 3/7 Glo assay?

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Kais Khudhair al Hadrawi

The presence of Green Fluorescent Protein (GFP) could potentially interfere with both absorbance measurements in an MTT assay and luminescence measurements in a Caspase 3/7 Glo assay, depending on the specifics of the assay protocols and the experimental setup. Here's how:

MTT Assay:In an MTT assay, the conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan by mitochondrial enzymes produces a colored product, which is quantified by measuring absorbance at a specific wavelength (usually around 570 nm). If GFP is present in the sample, its fluorescence emission spectrum could potentially overlap with the absorbance spectrum used for the MTT assay, leading to interference and inaccurate measurements. To minimize interference, you could consider: Background subtraction: Measure absorbance in wells without cells to account for any fluorescence from GFP. Use appropriate controls: Include control samples with known GFP expression levels to assess and correct for potential interference. Choose a different wavelength for absorbance measurement: Selecting a wavelength where GFP fluorescence is minimal or absent can help mitigate interference.

Caspase 3/7 Glo Assay:In a Caspase 3/7 Glo assay, the luminescence signal is generated by the cleavage of a substrate by active caspase 3/7 enzymes, resulting in the release of a luminescent compound. If GFP is present, its fluorescence could potentially contribute to the background signal, leading to elevated baseline luminescence readings and reduced assay sensitivity. To minimize interference, you could consider Background subtraction: Measure luminescence in wells without cells to account for any fluorescence from GFP. Use appropriate controls: Include control samples with known GFP expression levels to assess and correct for potential interference. Optimize assay conditions: Adjust assay parameters such as incubation time, substrate concentration, or cell density to maximize signal-to-noise ratio and minimize background luminescence.

In both cases, careful experimental design, proper controls, and optimization of assay conditions are crucial for accurate and reliable results, particularly when GFP or other fluorescent proteins are present in the samples.

Alaa Kaoud

Thank you for the explanation @ Kais Khudhair al Hadrawi.