Catalog #PS1001 (5 x 1ml per Unit)

FluoroStain™ Protein Fluorescent Staining Dye (Red, 1000X)

Description 
The FluoroStain Protein Fluorescent Staining Dye (Red, 1000X) is designed to substitute the common Coomassie Blue protein staining method, offering greater sensitivity and ease of operation. Unlike Coomassie Blue stain, the FluoroStain Protein Fluorescent Staining Dye binds to protein with high specificity, making destaining process an option rather than a requirement. With further reduction of background signals via destaining process, the FluoroStain is capable of achieving detection level parallel to silver staining without specialized imaging equipment (Figure 1), making it one of the most sensitive dyes available. In addition to its remarkable sensitivity, the FluoroStain Protein Fluorescent Staining Dye (Red, 1000X) brings a more reliable and safer user experience, since the stained gel can be visualized with blue-light illumination, avoiding the risk of skin/ eye damage caused by UV light. For best result, we suggest using B-BOX™ Blue Light LED epi-illuminator to visualize and analyze the gel stained with FluoroStain Protein Fluorescent Staining Dye (Red, 1000X). 

The FluoroStain Protein Fluorescent Staining Dye is compatible to the analysis of mass spectra (Fig. 2), i.e. LC-MS/MS, MALDI-TOF, etc.
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              Figure 1 
Comparison of FluoroStain Protein Fluorescent Staining Dye (Red, 1000X)
with Silver stain of a 2X serially diluted BSA sample.
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              Figure 3 
Excitation and emission spectrum of FluoroStain Protein Fluorescent Staining Dye (Red, 1000X).

Spectral Characteristics
When it is bound with bovine serum albumin (BSA), the fluorescent emission of FluoroStain Protein Fluorescent Staining Dye can be excited by UV and blue light sources, with excitation peaks around 369 and 517 nm and emission at 605 nm (Figure 3). In absence of BSA, FluoroStain Protein Fluorescent Staining Dye shows ignorable fluorescence as compared with protein-bound form, therefore giving a clear background for photographic analysis.
These spectral characteristics made this fluorescent dye compatible with a wide variety of gel reading facilities, including UV/ blue light epi- and transilluminator, argon laser and mercury-arc lamp excitation gel scanners.
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                       Figure 2
Compariosn of MALDI-TOF mass spectra of bovine serum albumin (BSA) stained with FluoroStain Protein Fluorescent Staining Dye (A) and with Coomassie Blue (B). BSA proteins are seperated on SDS-PAGE, stained with fluorescent dye or conventional Coomassie Blue, followed by trypsin digestion in gel, and then analyzed by MALDI-TOF. 
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Working Reagent Preparation
1:1000 dilution in 40% ethanol and 2% H₃PO₄

Storage 
-20°C   ≥ 12 months 
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Standard Protocol
1. Perform protein separation by SDS-PAGE.
2. Dilute the stock FluoroStainTM Protein Fluorescent Staining Dye (Red, 1000X) at a 1:1,000 ratio to a staining solution consisting of 40% EtOH, 2% H3PO4 in deionized water. The volume of staining solution should be at least 15 times of the gel’s volume (see Table 1).
3. Immerse the gel in the staining solution (1X) and incubate at room temperature for 2 to 24 h.  The quantity of staining solution needed is proportional to the gel size, e.g. 100 ml staining solution is an optimal condition for a 1.0 mm thick and 9 x 7 cm SDS-PAGE gel.  For an optimized sensitivity by minimization of the background signals, a quick rinse with deionized water before staining is suggested. Avoid immersing the gel in the SDS-PAGE running buffer before staining.  Use a plastic container. Glass containers are not recommended, as they absorbs much of the dye in the staining solution.  Protect the staining container from light by 6 covering it with aluminum foil or placing it in the dark.  No fixation procedure is required.  The staining solution shows better staining effect with fresh preparation.
4. Visualize or photograph the gel with a UV system or a blue-light illumination.  It is important to clean the surface of the epi-illuminator or the trans-illuminator with deionized water or 70% ethanol to avoid accumulation of dye, gel debris, and proteins on the surface.  The video cameras and CCD cameras have a different spectral response as compared with black-and-white print film and thus may not exhibit the same sensitivity.  If higher sensitivity and lower background is desired, destain the gel with destaining solution consisting of 7% EtOH, 2% H3PO4 in water.

Quick Protocol
1. Perform protein separation by SDS-PAGE.
2. Dilute the stock FluoroStainTM Protein Fluorescent Staining Dye (Red, 1000X) at a 1:1,000 ratio to a staining solution consisting of 40% EtOH, 2% H3PO4 in deionized water. The volume of staining solution should be at least 15 times of the gel’s volume (see Table 1).
3. Immerse the gel in a 1X staining solution of sufficient volume (Table 1), and heat by microwave oven until boiling*, followed by incubation at room temperature with a gentle shake for at least 30 min.
4. Visualize or photograph the gel with UV or blue-light illumination. 5. Destain with destaining solution consisting of 7% EtOH, 2% H3PO4 in deionized water for 30 min if necessary. *Caution: Handle the heated buffer with care. Avoid inhaling and contact with hot vapour. Proper protective wares are recommended.
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Table 1.
The suggested volume of staining solution.

Gel dimension     Gel volume     1X Staining
(1mm thick)                                 Solution  
9 cm × 7 cm           ≈ 6.5 ml           ≈ 100 ml
13 cm × 9 cm         ≈ 12 ml            ≈ 180 ml
16 cm × 16 cm       ≈ 26 ml            ≈ 390 ml
26 cm × 23 cm       ≈ 60 ml            ≈ 900 ml
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Notice and Caution Before Opening FluoroStain Vial:
Warm the vial to an ambient temperature to ensure that the fluorescent dye is thoroughly thawed and the solution is homogeneous. The stock solution should be handled with particular care because the solvent is known to facilitate the entry of organic molecules into tissues. 

Please dispose of the stain in accordance with local rules and regulations.