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ProStain™

accurate, fluorescent quantitation of cellular extracts

ProStain™ simplifies the determination of cell extract protein concentrations by providing highly sensitive detection reagents in a convenient and easy-to-use format. The fluorescent dye in ProStain is not effected by many commonly used buffer components such as detergent and salt (Figure 1), which can skew the results of other protein quantification systems. And, the spectral properties of ProStain change greatly after it is bound, effectively eliminating any background from unbound dye (Figure 2 and Table 1). These characteristics make the ProStain Kit a significant improvement over other detection systems.

Determining the protein concentration of cell extracts is an essential step in many modern cell biology procedures, such as Western blot and ELISA. Not surprisingly, there are numerous photometric and fluorescent methods for quantifying cell extract protein levels. Photometric methods have traditionally been based either on intrinsic absorbance at 280 nm or on reagent-based assays such as the Bradford and Lowry assays. However, these classical approaches tend to be time consuming, have limited sensitivity and may be influenced by the presence of substances that are commonly found in buffers. Fluorescent-based detection methods, while generally more sensitive, have also tended to suffer from limitations, such as the requirement for toxic co-factors, high rates of hydrolysis or unsuitable spectral properties. The ProStain Kit offers significant improvements in sensitivity, assay robustness and convenience when compared with existing methods.

Figure 1: Linear quantification in the presence of common buffer components.

Increasing amounts of BSA protein were quantified using the ProStain Protein Quantification Kit in the presence of a variety of buffer components that have been shown to effect other commonly used methods to quantify proteins.

Figure 2: Absorption and emission spectra of free vs. bound dye.

Normalized absorption and emission spectra of free (solid, purple lines) and conjugated dye (dotted, copper lines) in phosphate buffer of pH 7.2. As free dye and bound dye absorb at different wavelengths (612 nm vs. 503 nm) and the quantum yield of bound dye is 50-fold greater than that of free dye, background from free dye is effectively eliminated.

ProStain™ Advantages

A disadvantage of many protein quantification methods, such as the Bradford assay, is that the absorbance spectra of the free and conjugated forms of the dye partially overlap. This causes non-linear protein measurement, as the free dye is excited by the same wavelength of light used to excite the bound dye. In contrast, the free versus conjugated absorbance maxima of ProStain dye are separated by > 100 nm (Figure 2 and Table 1). In addition, nearly all free ProStain dye is hydrolyzed during the conjugation reaction, which improves the linearity and accuracy of the assay because excitation of the sample at 488 nm can only excite dye that is conjugated to protein. The small amount of free dye that remains is not excited at this wavelength, so background is completely eliminated. Finally, ProStain conjugation is fast and easy; simply resuspend the dye, add it to the wells of a microplate, then add a serial dilution of the BSA standard, along with your sample. After just 30 minutes, the fluorescence is read and the protein concentrations are easily quantified.

• Large Stokes shift & increased quantum yield of bound dye, and auto-hydrolysis of free dye, improve sensitivity and eliminate background
• Robust – limited effect from substances commonly found in cell lysis buffers
• Fast and simple procedure for cell extract protein concentration determination
• ProStain dye has only one reactive group, preventing crosslinking-related issues
• Supplied ready to use, eliminating the need for activation steps prior to conjugation

Contents & Storage

Each ProStain Kit supplies sufficient reagents for 1000 reactions. All reagents can be stored at 4°C and are guaranteed stable for 6 months when stored properly.