ProStain™ simplifies the determination of cell extract protein concentrations by providing highly sensitive detection reagents that are easy to use. The dye used in ProStain is resistant to many commonly found contaminants in cell lysis buffers such as detergent and salt, which limit the usefulness of other protein quantification systems. And, the spectral properties of ProStain change greatly after it is bound, effectively eliminating any background from unbound dye (see Properties tab). 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 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.
- 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.
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.
|Dye State||Absorption||Emission||ε L/(mol-cm)||Quantum Yield|
|Free dye||612 nm||665 nm||60,000||< 1%|
|Conjugated dye||503 nm||600 nm||24,000||50%|
|Table 1: Properties of ProStain dye.|
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.