SMCC-NH-CH2-triazole-PEG8-oxydiacetamide-Lys(MTT)-PAB

The MTT assay is a colorimetric endpoint assay for estimating viable cell number, cell growth, cytotoxicity, or cell activation in cultured mammalian cells. Living cells reduce the yellow tetrazolium salt MTT into purple/blue formazan, while dead cells do not generate the same signal; the resulting color can be quantified with a multiwell spectrophotometer. MTT reduction is commonly interpreted as a readout of metabolic activity that often correlates with viable cell number, but it should not be treated as a direct cell-counting method unless the assay is optimized for the cell type and experimental condition. Studies show that MTT reduction can involve mitochondrial and non-mitochondrial reducing systems, and formazan may accumulate in intracellular lipid droplets rather than simply marking mitochondria.

Gel electrophoresis is a method for separating biological macromolecules (such as nucleic acids or proteins) by forcing them through a gel matrix under an electric field.

Protein electrophoresis separates proteins in a polyacrylamide gel under an electric field; in SDS-PAGE, sodium dodecyl sulfate denatures proteins and gives them a broadly similar negative charge-to-mass ratio, so migration mainly reflects apparent molecular mass through the gel matrix. The readout is a stained protein band pattern: band position estimates apparent molecular mass using protein standards, band intensity reflects relative protein abundance within the linear range of staining/detection, and changes in band pattern can reflect protein expression, degradation, purification, or sample composition.

The following protocol uses the MTT colorimetric assay as a classic literature-established method for assessing cell viability/metabolic activity in cultured mammalian cells. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced by metabolically active cells to a colored formazan product; the amount of formazan is quantified spectrophotometrically and provides an indirect measure of metabolically active viable cells. Importantly, MTT reduction reflects cellular oxidoreductase/metabolic activity rather than an absolute direct count of living cells, so changes in cellular metabolism can alter the signal independently of cell number.