Advanced Ruthenium (II) tris (bathophenantroline disulfonate) staining: Western blotting and sequential silver staining
Andreas Lamanda1 (Dr. phil. nat.),Melek Dilek Turgut2 (Dr. med. dent.)
In this study, the compatibility of advanced RuBPS polyacrylamide gel staining with blotting techniques and sequential silver staining was described. The higher dye concentration was assumed to lead to incompatibilities with the subsequent analytical methods. However, this study demonstrated that this assumption was arbitrary. RuBPS pre-stained proteins were transferred from a 2-D gel to a stack of nitrocellulose membranes producing up to nine replica of the original spot pattern without the need of additional membrane staining steps. The method further allowed the transfer of a RuBPS pre-stained 18 kD protein without disturbing the sequential antibody detection, in western blotting. Finally, sequential silver staining of RuBPS stained 2-D gels resulted in virtually better staining quality and increased spot count compared to SYPRO Ruby staining followed by silverstaining.
In recent years, 2-D electrophoresis has been accepted as a standard procedure to separate complex protein mixtures in proteome studies. Protein visualisation by fluorescence detection has become a firmly established and widely used method [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15] in proteomic analysis and a crucial step in protein expression profiling. For protein detection, it is advantageous to use fluorescent labels containing chromophores which have longer excitation and emission wavelength than the aromatic amino acids. The dyes used for this important step should combine attributes like good signal to background ratio (contrast), broad linear dynamic range, broad application range, photo stability and compatibility to protein identification techniques, e.g. mass spectrometry (MS) or western blotting.
Originally, the transition metal complex, Ruthenium (II) tris (4,7-diphenyl-1,10-phenantrolin disulfonate) also termed as Ruthenium (II) tris (bathophentroline disulfonate) was synthesized as a precursor for a dye that was used as a non-radioactive label for oligo nucleotides . Later, Rabilloud et al.  used RuBPS as a fluorescent label for protein detection in polyacrylamide gels. The fact that RuBPS is not only easy to synthesize but also easy to handle, induced further developments in this field. Lamanda et al.  improved the RuBPS staining protocol by selectively destaining the polyacrylamide matrix while the protein content remained tinctured. This new technique entailed a variety of advantages like strong signals, ameliorated signal to background ratio, better linearity and advanced baseline resolution. However, the question if the advanced staining method interferes with subsequent analytical methods still remains to be answered.
The aim of this work was to demonstrate the compatibility of the advanced RuBPS staining method with subsequent protein identification using MALDI-TOF MS in comparison with a standard method applying coomassie blue staining. Additionally, it was aimed to demonstrate, for the first time, the blotting of pre-RuBPS-stained proteins on nitrocellulose and/or PVDF membranes followed by immunostaining (western blotting) and to compare sequential advanced RuPBS-silver staining with sequential SYPRO Ruby-silver staining.
E. coli cells MC 4100 were grown in batch cultures on LB medium to OD600=0.8. Cell harvesting and disruption were done according to the standard procedures.
Electrophoretic separation was performed on a Mini-PROTEAN® 3 Cell (BioRad, Rheinach, Switzerland) using a 17.5 % polyacrylamide gel as previously described .
E.coli proteins were prepared and separated as previously described  with the change that 240 g E. coli total protein were loaded per IPG strip.
The gels were stained with Ruthenium II tris-bathophenantroline disulfonate (RuBPS)as previously described . For sequential staining, RuBPS pre-stained gels were silver stained. SYPRO Ruby was used as recommended by the manufacturer and sequentially silver stained as previously described .
Silver stained gels were scanned on a flatbed scanner (HP Deskscan, software DeskScanII V2.3) with the following scanning parameters: 300x300 dpi, 8 bit black and white picture (256 grey shades, twice sharpened)
, contrast of 125 and brightness of 125. RuBPS stained gels, membranes and western blots were scanned with a scanner (Fuji FLA-3000 with software BASReader v3.01) using the following parameters: resolution of 50 mm, 16 bit picture (65’536 grey shades), sensitivity of 1000, excitation wavelength of 473 nm and detection filter of O580. Images were processed with the advanced image data analyzer software (AIDA, v3.10, v4.1).
RuBPS stained 2-D gels, PVDF and nitrocellulose membranes were equilibrated in a blotting buffer (50 mM boric acid, pH 9.0) for 30 min. Stained protein spots were electrophoretically transferred either to a nitrocellulose membrane (or a stack of 8 membranes) for 45 min. at 200 mA and 500 V or to a PVDF membrane for 90 min. at 200 mA and 500 V under cooling. Membranes were washed twice with TBS (10 mM Tris/HCl pH 8.0, 150 mM NaCl) and saturated with TBS containing 1% w/v milk powder for 10 min. After rinsing with TBS, the membranes were incubated overnight with a rabbit anti IIAGlc antibody (1:5000) in TBS containing 0.5% w/v BSA. The membranes were then rinsed in TBS and incubated with an anti rabbit IgG peroxidase coupled antibody (1:5000) in TBS, 0.5% w/v BSA for 2 h. After the reaction stopped, the membrane was washed twice in 0.5% TBS for 3 min. and then treated with peroxidase staining solution (TBS, 6% v/v chloro-1-naphthol 0.3% w/v in MeOH, 0.002% v/v H2O2 30%). After 2 to 5 min., the antibody bound proteins became visible and the reaction was stopped by incubating the membrane in 0.5% TBS.
Co-blotting of RuBPS stained Protein on nitrocellulose and/or PVDF membranes
It was tested whether RuBPS-prestained proteins were electro transferable from a gel to a membrane under protein fixing conditions. One mg RuBPS stained BSA per band was transferred from a gel on a double layer of membranes (Nitrocellulose/PVDF) after electrophoresis (Fig. 2). The nitrocellulose layer was covered with 8 of the 12 BSA lanes (Fig. 2A) to test if a direct transfer to PVDF (Fig. 2B) and also an indirect transfer through a nitrocellulose membrane were possible. After the transfer, fluorescent bands were detectable on both membranes corresponding to the original protein locations in the gel, implying that RuBPS or RuBPS-protein complexes were transferred from the gel to the membranes.
To expand the method, 100 mg of rat hypocampus total protein preparation were separated by 2-D electrophoresis and stained as previously described . A gel piece 5´7 cm in seize was cut out from the gel. The protein spot pattern of this gel piece (Figure 3, panel G) was transferred to a stack of 8 nitrocellulose membranes topped by a PVDF membrane. The gel contained 14 protein spots which were fully detectable in all 9 membranes indicating that the protein-dye complexes were moved through the membrane stack (Fig 3 Panel G, 1, 5 and 9). The spot intensities linearly increased from the innermost to the top membrane. However, it was impossible to conclude that all the detected spots on the membrane were composed of protein and dye molecules. Therefore, an entire 2-D gel of E. coli whole cell extract was transferred onto a PVDF membrane in order to detect IIA glucose (IIAGlc), a kinase of the bacterial phosphoenolpyruvate dependant phosphotransferase system, selectively by Western blotting. By this way, it was possible to control if protein was successfully transferred and the dye did not interfere with the antibody used for detection. The exact pI and Mr coordinates in the gel of the monomeric protein were confirmed by mass spectrometric identification. IIAGlc produced a single spot at 18 kD and pH 4.2. The black spot of IIAGlc was located in the RuBPS stained gel (Fig 3A) as well as on the membrane (Fig 3B). After immunodetection, a single black spot appeared on the membrane at the coordinates of IIAGlc by visual inspection. At exactly the same location the rescanned blot exhibited the strong white landmark spot of IIAGlc.
Sequential silver staining of a RuBPS stained gel.
Two 2-D gels of E. coli total cell proteins were prepared. The first one was stained with RuBPS (Fig. 4A), while the second one was stained with SYPRO Ruby (Fig. 4C). The RuBPS stained gel featured 842 protein spots (Fig. 4A) whereas the SYPRO Ruby stained gel had 574 spots (Fig 4C). Both gels were sequentially stained with silver nitrate. In the RuBPS pre-stained gel, the detected number of spots was found to be 1064 (Fig. 4B). The doubly stained gel had an almost clear background. In the SYPRO Ruby pre-stained gel the spot count was found to be 479 (Fig. 5D) after silver staining, and the gel exhibited a dull brownish background.
The present study aimed to demonstrate the compatibility with Western blotting and sequential silver nitrate staining. A perfect method should combine a sensitive in-gel visualization followed by a sequential electrophoretic transfer of protein-dye complexes to membranes and allow for the combination with immunodetection of specific proteins. At present, some reports in the literature are found to be close to the ideal conception. Steinberg et al.  were able to demonstrate this type of methodical coupling for SYPRO Tangerine. The drawback of their method was the dependency of the non protein fixing SYPRO Tangerine dye as well as the salt concentration linked to a relatively low sensitivity. The same author presented a method to transfer SYPRO Orange pre-stained proteins to nitrocellulose membranes. However, the method was restricted to non-denaturing conditions . A technique to transfer proteins to several membranes was also reported but the major drawback of it was to operate only for unstained protein .
The new presented method in this study, however, combines electrotransfer of RuBPS pre-stained proteins from 1-D and 2-D gels to up to 9 membranes (Fig. 2, Fig 3) followed by immunodetection (Fig 4, Panel C). The evidence that the dye-protein complex was transferred from gel to membrane proved by the identification of IIAGlc by immunodetection. The location of IIAGlc on the membrane was labelled by a black spot caused by the reaction of the peroxidase coupled antibody. The black colour absorbed the laser light of 473 nm used for excitation (Fig 4 Panel C). This effect became visible by the appearance of IIAGlc as a white landmark spot in a bulk of black coloured surrounding protein spots on the membrane (Fig 4, Panel C).
If fluorescent stained gels have to be conserved for documentation, for future analysis or location of landmark protein spots, they need to be stained with CBB or silver followed by drying on a filter paper. If necessary, the spot patterns from dried 2-D gels can be linked to the spot patterns of fluorescent labeled gels. The method presented in this study was concluded to be more useful since some proteins might escape from identification when they had been solely stained with Ruthenium dyes [24,25]. Ideally, a sensitive silver staining procedure should be sequentially applicable to fluorescent staining without causing an additional background. Miura et al.  used a fluorescently stained gel with SYPRO Orange for subsequent staining with silver. However, the resulting staining quality was documented by a relatively high background.
The combination of RuBPS and silverstaining did not cause an additional background or any other side effect (Fig 4, Panel B). The 25% increased spot number after silverstaining corresponds well with the higher sensitivity (1 ng protein) of silver staining compared to RuBPS staining (8 ng Protein) . A lowered sensitivity for silverstaining was observed in a pre-SYPRO Ruby stained gel where the spot count drops by 13% from 574 (Fig 4., Panel C) to 479 (Fig 4, Panel D). This drop of spot number may be explained by the blurring effect produced in combination with the high background arising from one of the components of the SYPRO staining solution. The observed blurring eventually hampers the delicate automated software aided spot detection and may further drop the even low spot number.
Currently RuBPS is the only dye that can be used in mass spectrometric protein identification, Western blotting and sequential silverstaining. The results of the present study pronounce the high possibility of using RuBPS on several stages in 2-D electrophoresis.
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