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Three new fluorescent protein stains, LUCY-506, LUCY-565 and LUCY-569 have been developed. These dyes are suitable for staining proteins on polyacrylamide gels (both 1D- and 2D-Electrophoresis) and offer a reliable, convenient and economic alternative to current silver staining techniques or existing fluorescent staining methods for the detection of minute amounts of protein.
In general the staining procedures are easy, fast and robust. The standard procedure is a post-electrophoretic stain without fixation-step, which is completed after 60min. Using a modified staining protocol, it is also possible to stain a gel with LUCY-565 and perform a western-blot afterwards. Native gels can be visualized by rinsing the gel in SDS immediately after the run, but before staining it. Table 1 summarizes the suitability and performance of these dyes for different staining procedures.
Several devices can be used for the detection, e.g. illuminating the gel on a transilluminator (Dark-Reader, UV-Screen) and imaging the gel using a CCD- or Polaroid-Camera. Alternatively a laser scanner can be employed, using the corresponding excitation and filter settings. Table 2 shows an overview over the applicable systems for illumination.
The profile of each of the dyes has been established: LUCY-506 shows the highest sensitivity, LUCY-565 allows neutral staining (e.g. before Western blotting) and LUCY-569 excels by a linear response over an extraordinary broad linear dynamic range.
Figure 1: Staining of 2-D-minigels: 10ug E.Coli-extract, 7cm IPG-strips pH 3-10, Novex Tris-Glycine gels 4-20%
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LUCY-506 , 0,1% SDS laser scanner,
λex 473 / filter 520
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SYPRO Ruby , 0,1% SDS laser scanner,
λex 473 / filter 580
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Standard procedure |
Prestaining procedure |
Stain after fixation |
Native PAGE stain |
Neutral stain before WB |
| Post-electrophoretic stain in HOAc; time required: 1h |
Dye in cathode-running-buffer with subsequent destaining in HOAc; time required: 15-60min |
TCA-fixation; SDS-rinse; stain in NaOAc; time required: 1h45min |
Run gel SDS-free; rinse gel in SDS; then std procedure; time required: 1h30min |
Postelectrophoretic stain in water; time required: 1h; continue with Western-Blot transfer |
| Lucy-506 |
+++
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++
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+
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+++
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-
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| Lucy-565 |
+
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+
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++
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+++
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+++
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| Lucy-569 |
+++
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++
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++
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+++
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-
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Table 1: Suitability and performance of Lucy stains for various staining procedures.
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LUCY-506
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LUCY-565
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LUCY-569
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| imaging device |
illumination |
filter |
sensitivity |
filter |
sensitivity |
filter |
sensitivity |
| Polaroid camera |
UV screen
(λ max. ~ 310 nm) |
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Dark Reader®
(λ max. ~ 450 nm) |
orange filter |
+
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| CCD camera |
UV screen
(λ max. ~ 310 nm) |
590 nm band pass |
-
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590 nm band pass |
++
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590 nm band pass |
+++
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Dark Reader®
(λ max. ~ 450 nm) |
590 nm band pass |
+++
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590 nm band pass |
+
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590 nm band pass |
+
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| amber filter |
+
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amber filter |
-
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amber filter |
-
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| laser scanner |
473 nm laser |
520 nm cut off |
+++
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520 nm cut off |
-
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520 nm cut off |
-
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| 532 nm laser |
580 nm cut off |
-
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580 nm cut off |
+++
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580 nm cut off |
++
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| 630 nm laser |
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Table 2 : Imaging devices, methods and performance
The detection limits for the 3 dyes ranged between 3-10 ng/band in 1D-PAGE. Fig.1 shows a comparison between LUCY-506 and Sypro Ruby in 2D electrophoresis. Comparisons to current state-of-the-art dyes were drawn under different experimental conditions. In-gel digested proteins can be analysed by mass spectrometry
Linearity
Lucy dyes generally provide a large range of linearity. Linearity was given up to 1000-6000 ng/band and is therefore larger than for silver stains (White et al., Electrophoresis 2004; 25(17); 3048-3054), coomassie blue or other fluorescence dyes.
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| Figure 2: Dynamic range of Lucy stains in comparison to other staining methods. |
For other staining reagents please click here
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