The Thermo Scientific Pierce Coomassie Plus Protein Assay is a high-performance Bradford assay reagent one of the fastest,

, simplest and most popular protein quantitation methods available. The Pierce Coomassie Plus Assay Reagent is a single, ready-to-use solution for measuring protein concentration. Simply add the reagent to equal volumes of samples and standards, mix and then measure the absorbance at 595nm. The assay costs only pennies per sample and can be performed in either test tube or microplate format. The Pierce Coomassie Plus Assay Reagent provides increased linearity of response and only half the protein-to-protein variability of other commercial Bradford assay formulations. Highlights: Colorimetric measure with a standard spectrophotometer or plate reader (595nm) Easy to use single reagent; no working reagent preparation

How the Coomassie Plus (Bradford) Assay Detects Protein: Use of coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of total protein was first described by Dr. Marion Bradford in 1976. In the acidic environment of the reagent, protein binds to the coomassie dye. This results in a spectral shift from the reddish/brown form of the dye (absorbance maximum at 465nm) to the blue form of the dye (absorbance maximum at 610nm). The difference between the two forms of the dye is greatest at 595nm, so that is the optimal wavelength to measure the blue color from the coomassie dye-protein complex. If desired, the blue color can be measured at any wavelength between 575nm and 615nm. At the two extremes (575nm and 615nm) there is a loss of about 10% in the measured amount of color (absorbance) compared to that obtained at 595nm. Development of color in coomassie dye-based (Bradford) protein assays has been associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the protein. Van der Waals forces and hydrophobic interactions also participate in the binding of the dye by protein. The number of Coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein. Free amino acids, peptides and low molecular weight proteins do not produce color with coomassie dye reagents. In general, the mass of a peptide or protein must be at least 3,000 daltons to be assayed with this reagent. The assay is performed at room temperature and no special equipment is required. Simply add the sample to the tube containing reagent and the resultant blue color is measured at 595nm following a short room-temperature incubation. The coomassie dye containing protein assay is compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents encountered in protein samples.

Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered.[12] The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien.[13] This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm, with the peak emission kept at 509 nm. This matched the spectral characteristics of commonly available FITC filter sets, increasing the practicality of use by the general researcher. A 37 C folding efficiency (F64L) point mutant to this scaffold yielding enhanced GFP (EGFP) was discovered in 1995 by the lab of Ole Thastrup.[14] EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ) of 55,000 M1cm1.[15] The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as QY, is 33,000 M1cm1. Superfolder

GFP, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.[16] Many other mutations have been made, including color mutants; in particular, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet). BFP derivatives (except mKalama1) contain the Y66H substitution. The critical mutation in cyan derivatives is the Y66W substitution, which causes the chromophore to form with an indole rather than phenol component. Several additional compensatory mutations in the surrounding barrel are required to restore brightness to this modified chromophore due to the increased bulk of the indole group. The red-shifted wavelength of the YFP derivatives is accomplished by the T203Y mutation and is due to -electron stacking interactions between the substituted tyrosine residue and the chromophore.[2] These two classes of spectral variants are often employed for Frster resonance energy transfer (FRET) experiments. Genetically-encoded FRET reporters sensitive to cell signaling molecules, such as calcium or glutamate, protein phosphorylation state, protein complementation, receptor dimerization, and other processes provide highly specific optical readouts of cell activity in real time. Semirational mutagenesis of a number of residues led to pH-sensitive mutants known as pHluorins, and later super-ecliptic pHluorins. By exploiting the rapid change in pH upon synaptic vesicle fusion, pHluorins tagged to synaptobrevin have been used to visualize synaptic activity in neurons.[17]

mice expressing GFP under UV light Redox sensitive versions of GFP (roGFP) were engineered by introduction of cysteines into the beta barrel structure. The redox state of the cysteines determines the fluorescent properties of roGFP.[18]