ProStain™ Protein Quantification Kit

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 methods 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 contaminating substances. 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.

ProStain™ advantages

• Large Stokes shift and increased quantum yield of bound dye improve sensitivity and eliminate background
• Robust – limited effect from contaminating substances 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

Figure 1: Standard curves of BSA in various contaminants.
Increasing amounts of BSA protein were quantified using the ProStain Protein Quantification Kit in the presence of a variety of contaminants, demonstrating that ProStain is not influenced by these compounds.

The background of ProStain is extremely low because its absorption max shifts by over 100 nm after the dye is bound, and because bound dye has a 50-fold greater quantum yield than free dye (Figure 1, Table 1).

Figure 1: Absorption and emission spectra of free vs. bound ProStain dye.
Normalized absorption and emission spectra of free (solid, purple lines) and conjugated ProStain 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.

The ProStain™ Protein Quantification Kit Assay has been described and used in the following publications:

“Monitoring of endothelial cell activation in experimental sepsis with a two-step cell culture model” by Schildberger et al. (2010) Innate Immunity 16(5):278-287.
 
“Non-apoptotic Fas-induced regulation of cytokines in undifferentiated and decidualized human endometrial stromal cells depends on caspase-activity” by Fluhr et al. (2011) Mol. Hum. Reprod. 17(2):127-134.
 
“Determination of drug toxicity using 3D spheroids constructed from an immortal human hepatocyte cell line” by Fey & Wrzesinski (2012) Toxicol. Sci. 127(2):403-411.

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.