Estimation of submicrogram quantities of protein using the dye eosin Y

Eosin B is used to estimate proteins above 1 microg/ml concentration [Waheed AA, Gupta, PD. Anal. Biochem. 1996:233:249-256; Waheed AA, Gupta PD. J. Biochem. Biophys. Meth. 1996;33:187-196]. In the present report we describe a method for estimating submicrogram quantities of proteins using the dye eosin Y. The increase in sensitivity of this assay is approximately two fold under optimal assay condition. The optimum concentration of eosin Y and citric acid for submicrogram assay is 0.01 and 0.05%; (final concentration) respectively. The protein-dye complex formation is completed within 2 min and its absorbance is stable up to 60 main with a variation of +/-4.0%. The interference due to sugars, reducing agents, glycerol and some neutral detergents like Triton X-100, NP-40 and Tween-20 is less than 12% whereas Brij-35, ethanol, acetone and chelators like EGTA and EDTA suppress the absorbance by about 12-18%. However, basic buffers like Tris, urea, CHAPS and NaN, interfere with the formation of the protein-dye complex. The increase in absorbance of protein-eosin Y complex compared to that of protein-eosin B complex is due to the higher extinction co-efficient of eosin Y compared to eosin B.