Accurately determine protein concentration in the presence of 2% SDS, 1% DTT, 8 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte, and 2% IPG Buffer. Quantitatively precipitates proteins while leaving interfering substances behind. Linear response in the range of NA to 5NA μg protein, with recommended sample volumes of 1 to 5NA μl. Common spectrophotometric methods of quantitating protein rely either on Coomassie dye binding or proteincatalyzed reduction of cupric (Cu2+) ion to cuprous (Cu+) ion. Dyebinding assays cannot be used in the presence of any reagent that also binds the dye. This includes carrier ampholytes and detergents such as PlusOne CHAPS, SDS, and Triton X1NANA. Assays that depend on the reduction of cupric ion cannot be used in the presence of reductants such as DTT, or in the presence of reagents that form complexes with cupric ion, such as thiourea or EDTA. The 2D Quant Kit procedure works by quantitatively precipitating proteins while leaving interfering substances behind. The assay is based on the specific binding of copper ions to protein. Precipitated proteins are resuspended in a coppercontaining solution and unbound copper is measured with a colorimetric agent. The absorbance at 48NA nm is inversely related to the protein concentration. The assay has a linear response to protein in the range of NA to 5NA μg and recommended sample volume is 1 to 5NA μl. Reagents tested for compatibility with the 2D Quant Kit Compound Tested: Concentration
SDS : 2% (w/v)
CHAPS : 4% (w/v)
Triton X1NANA : 1% (w/v)
Pharmalyte pH 31NA : 2% (v/v)
IPG Buffer pH 31NA NL : 2% (v/v)
Tris : 5NA mM
EDTA : 1NA mM
DTT : 1% (65 mM)
2Mercaptoethanol : 2% (v/v)
Urea : 8 M
Thiorea : 2 M
Glycerol : 3NA% (w/v)
