Two Additives in Ruthenium II tris (bathophenantroline disulfonate) protein staining
Andreas Lamanda (Dr. phil. nat.) and Stefan Reber (Dr. sc., dipl. ing. FH)
Staining with Ruthenium (II) tris (bathophenantroline disulfonate) is gaining popularity in one- and two dimensional electrophoresis technique. In this study, two new methods, 1,3-Didecyl-2-methyl-imidazolium chloride and ultrasound applications, as additives for RuBPS stained gels were described. 1 and 2-D gels were incubated in a solution of 0.1% 1,3-Didecyl-2-methyl-imidazolium chloride or treated with ultrasound. The cationic surfactant DMC enhanced the fluorescence of low abundant RuBPS stained proteins by up to 4.5 times and hence lowered the detection limit of RuBPS from 8 ng to 2 ng protein. By contrast, the fluorescence of high abundant proteins was almost totally quenched after DMC treatment. In adition, it was observed that the background of a RuBPS stained polyacrylamide gels can be uniformly reduced 0.05% of the initial value by sonication. The consequence was a negative relative signal reduction that became measurable as net signal amplification, for protein loads up to 250 ng. The two additives, 1,3-Didecyl-2-methyl-imidazolium and ultrasound, broaden the application range of RuBPS staining.
Keywords: 1,3-Didecyl-2-methyl-imidazolium chloride, cationic tenside, gel sonication, Ruthenium II tris (bathophenantroline disulfonate), RuBPS
When a fluorophore absorbs a photon of the excitation energy (ex) Eex=hc/λex, one of its electrons is shifted to an excited singlet state. The return of this electron to a ground-state electron with opposite spin orientation is spin-allowed and occurs by the emission (em) of a photon of the energy Eem=hc/λem. In this situation, the emission wavelength is longer than the excitation wavelength, which is known as Stokes’ shift. The process of irradiative electron relaxation is termed as fluorescence .
A charge transfer complex with bidentate nitrogen donors whichs’ excitation follows the description above is Ruthenium (II) tris (4,7-diphenyl-1,10-phenantrolin disulfonate), also termed as Ruthenium (II) tris (bathophentroline disulfonate) . RuBPS was first synthesized by Bannwarth et al.  with the aim to create a non-radioactive label for oligo nucleotides. Today, although RuBPS is not being used for DNA labeling, it is used for post-electrophoretic protein detection in polyacrylamide gels, with a sensitivity of 8 ng . During the last 6 years, RuBPS has regained its popularity among the researchers [05,06,07,08,09,10,11,12,13,14,15,16]. When it is used for protein detection, RuBPS forms a stable complex with proteins through electrostatic interaction. RuBPS is usually excited with UV light of 300 nm or alternatively with laser light of 450 to 490 nm. Fluorescence is detected at 610 nm with a photomultiplier or a CCD camera.
Up to our knowledge, there are no procedures or additives that enhance or reduce the fluorescence of RuBPS stained proteins in polyacrylamide gels. The aim of this article was therefore to introduce two new methods, with respect to (i) fuorescence enhancement for low abundant proteins by applying the cationic surfactant 1,3-Didecyl-2-methyl-imidazolium chloride, and (ii) reduction of background fluorescence of RuBPS stained gels by ultrasonication.
2. Experimental Procedures
2.1. 1-D and 2-D electrophoresis
Sample preparation, electrophoretic separation and two fold serial dilution of carbonic anhydrase was performed as described by Lamada et al. .
2.2. Modified RuBPS staining
RuBPS was prepared as described by Rabilloud . The gel was placed in 50 ml of 40% Ethanol/10% acetic acid containing 1 mM RuBPS for 1 hour. After 20 min. of destaining in 40% Ethanol/10% acetic acid, the gel was washed for 10 min. in water and then scanned.
2.3. Gel treatment with 1,3-Didecyl-2-methyl-imidazolium chloride
The RuBPS stained gels were incubated in a solution of 0.1% (25 mM) 1,3-Didecyl-2-methy-limidazolium chloride (C24H47ClN2 m=399 g/mol, Fluka 36757) in water for 20 min. Alternatively, gels were first incubated in water pH 11 for 20 min. The pH was confirmed frequently and readjusted to 11 with NaOH 1 mol/l, if required.
2.4. Gel sonication
The gel was placed in 2 l water within a table top ultrasonifier (model TUC-150A de Luxe, Wohlen, Switzerland) and ultra-sonified at 35 kHz for 60 min. Sonication was interrupted after 2 and 30 min. for protein detection.
2.5. Gel imaging
RuBP stained gels were scanned with an Amersham Storm 860 scanner (Amersham Bioscience, Freiburg, Germany). Images were processed with the advanced image data analyzer software (AIDA, v4.10).
2.6. Protein identification
Proteins were identified by matching the 2-D reference maps published at the Swiss 2-D PAGE Server (www.expasy.org/ch2d/).
3.1 DMC treatment
A RuBPS stained 0.75 mm thick 1-D gel containing a two fold serial dilution of carbonic anhydrase was stained with RuBPS  and incubated in 0.1% 1,3-Didecyl-2-methyl-imidazolium chloride. Another RuBPS stained gel was incubated in water at pH 11 prior to incubation in 0.1% 1,3-Didecyl-2-methyl-imidazolium chloride. Evaluation of the protein band intensities revealed that RuBPS stained carbonic anhydrase showed a typical linear dependence between fluorescence and the amount of protein that has been described earlier . Treatment with 1,3-Didecyl-2-methyl-imidazolium chloride did not change the dimensions of the gel but lead to a reduction of the 1000 and 2000 ng carbonic anhydrase signals to 0.5 and 0.6 % of the original intensity, respectively (Fig. 1). The signals corresponding to the 125 ng and 250 ng bands were quenched to 24 and 58% of their original intensitiy, respectively. The signals corresponding to the 4 to 67 ng DMC treated bands had an almost uniform fluorescence of 2400 ± 120 LAU. Additional DMC caused a doubling of fluorescence in the 8 ng band compared to RuBPS staining alone. The signals corresponding to the 2 and 4 ng bands became detectable in contrary to RuBPS staining (Fig 1). The background of this gel was 3.75 times enhanced.
Sequential incubation in water at pH 11 and 0.1% DMC did not improve sensitivity of DMC treatment. In this technique, similar to the DMC treatment the fluorescence of the 1000 and 2000 ng bands was quenched to 0.6 and 0.5 % of the initial fluorescence. The fluorescence of the 500 ng band was reduced to 6% of the initial value. In the 250 ng band, the fluorescence was enhanced 1.3 times. The signals corresponding to the 125 and 67 ng bands also exhibited enhanced fluorescence (1.1 and 1.3 times) respectively. The remaining (8 to 32 ng) bands all showed reduced fluorescence values. The background was found to be amplified 5.75 times.
For further investigations, 30 mg of total E.coli total cell protein were separated by 2-D PAGE. 450 RuBPS stained proteins contained an average protein load of 60 ng per spot. The sequential DMC treatment did not modify the dimension of the 1.5 mm thick gel. In Figure 2, the gel region from 10 to 25 kD and pH 5 to 5.6 was shown in panel A (RuBPS stained), panel B (RuBPS-DMC treated) and panel C (silver stained). There were 54 spots detected in the silver stained gel compared to 52 spots in the corresponding part of the DMC treated gel and 40 in the RuBPS stained gel. The coordinates of 14 low abundant proteins which were undetectable by RuBPS were marked with a white circle in panel A. They became detectable after DMC treatment (panel B) as demonstrated for peptide deformylase (spot 17) and GroES (spot 44). Fluorescence quenching was detected for high abundant proteins like alkyl-hydroperoxide-reductase subunit C (spot 3), superoxide-dismutase (spot 7), ATP-dependent-Clp-protease-proteolytic subunit (spot 24), alkaline-shock protein (spot 34), and nucleoside-diphosphate-kinase (spot 46).
The spot volumes of a 52 pixel area from the centre of 200 RuBPS stained spots from the 2-D gel were matched to their RuBPS-DMC treated counterparts and evaluated for a mathematical coherence. The RuBPS-DMC treated spot volumes (panel B) were linked to the corresponding RuBPS spot volumes (panel A) by the exponential decay function (I) (panel D):
VB(xy) = VA(xy)*12.586*e(-4*VA(xy)/1000) (I)
VAxy was the numerical integral of all pixel values from the 52 pixel spot area with coordinates x (isoelectric point) and y (mass) in the RuBPS stained gel and VBxy the analogue value from the corresponding spot in a RuBPS-DMC treated gel. The uniform background was measured at 200 locations before and after DMC treatment and found to be 3.5 times increased compared to RuBPS staining alone. Correlation between the spot intensity derived after DMC treatment and parameters such as the isoelectric point, the molecular mass, or the content of arginine and lysine residues of 25 identified proteins were investigated but no such correlations were determined.
A 0.75 mm thick polyacrylamide gel containing a two-fold serial dilution of carbonic anhydrase was stained according to an accelerated RuBPS staining procedure and sequentially ultrasonified (Fig. 3). 2 min. of sonication in water caused a 25% extension of the total gel area and hence the protein band areas. Therefore, a 45 pixel area of each band before sonication was compared to the corresponding 25% extended area (56 pixels) after sonication by 2-D densitometry. The same procedure was applied to the background measurements. In the center of Figure 3, lanes containing 8 ng to 518 protein before (upper row) and after sonication (lower row) are shown. A plot of the measured band intensities before and after sonication without subtraction of the background was demonstrated in panel A. The unsonified bands reflected a linear dependence between fluorescence and amount of protein (grey bars). After sonication (black bars), the intensity of bands from 32 to 518 ng was in the range of 4500 ± 160 LAU.
Signals were reduced to 87, 75 and 63% for 130, 259 and 518 ng protein, respectively. The 32 and 16 ng band intensities were found to be little increased (1%) compared to the original values. The signal corresponding to the 8 and 4 ng bands were reduced to 75 and 72 % of their initial values, respectively. The background measured at 30 locations was uniformly reduced to 68% of the initial value.
Figure 3, Panel B shows a plot the band intensities before and after sonication with background subtraction. After sonication, the intensity of the 518 ng band was reduced to 60% of the initial value. The intensity of the 259 ng band was reduced to 80% whereas the band containing 130 ng was intensified to 1.1 times. The signal corresponding to the 64 and 32 ng bands had a 1.7 times increased intensity which was the strongest measurable intensification. The signal corresponding to the 16 and 8 ng bands showed a 1.2 stronger intensity after sonication.
The procedure was also tested on a 2-D gel. 80 mg of total human serum were separated by 2-D electrophoresis, stained with RuBPS , and sonicated for 60 min. with an interruption for a scan after 30 min. The 1.5 mm thick 2-D gel grew 25% in its dimensions. A section of the gel from Mr 30-60 kD and pI 4.7-5.5 containing 6 strong spot trains was shown in Figure 4. The total spot count was 75. Spot trains were identified as isoforms of: 1) alpha-1-antitrypsin (6 spots, Swiss-Prot number P01009), 2) antithrombin-III (3 spots, P1008), 3) fibrinogen gamma chain, (3 spots, P02679), 4) haptoglobin (8 spots, P00737), 5) clusterin, (5 spots, P10909), and 6) transthyretin (3 spots, P02766). Spot intensities were determined immediately after RuBPS staining, after 30 min. as well as after 60 min. of sonication. The spot area was increased 1.5 times after 30 min. and 1.65 times after 60 min. of sonication. As the gel and spot area grew, 2-D densitometry was performed in the centre of each spot, in a 10 mm2 area. There was an average of 1.48 ± 0.3 times spot intensity enhancement after 30 min. and 1.64 ± 0.4 after 60 min. of sonication. Spot volumes of initially-strong spots like alpha-1-antitrypsin (spot train 1, 9500 LAU) was 1.38 times intensified after 30 min. and 1.43 times after 60 min. Weak spots like the 5 spots of clusterin (spot train 5, 3500 LAU) were intensified 1.5 times after 30 min. and 1.8 times after 60 min. The background measured at 200 locations was reduced to 1/36 after 30 min. and to 1/544 after 60 min. of sonication. The initial intensities of the 75 assayed spots were plotted against the amplification factor after sonication and subjected to a third degree polynomial fit (Fig. 5). The highest net signal amplification after 30 min. was 1.6 to 1.8 times higher than the original fluorescence after sonication. After 60 min. of sonication, this optimum was observed to shift to weaker spots with volumes ranging from 600 to 3500 LAU, exhibiting an 1.8 to 1.9 times amplification. 4, initially very weak proteins spots vanished after 60 min. of sonication but sequential silverstaining showed that all 75 proteins were present in the gel after sonication. Sonication did not interfere with sequential protein identification by peptide mass fingerprinting (results were not shown).
It has already been successfully demonstrated by Factor and Xu [17,18] that fluorescence can be influenced by surfactants. The cationic surfactant 1,3-Didecyl-2-methyl-imidazolium chloride was used to create a protein-tenside complex containing positive charged imidazolium groups. DMC enhanced the fluorescence of low abundant RuBPS stained proteins in 1-D and 2-D gels (2 to 8 ng). As the sulfonate residues of RuBPS are negatively charged, a greater number of RuBPS molecules may bind to the positive charged protein-tenside complex, compared to an SDS coated negatively-charged protein which is present after electrophoresis. The increased number of bound RuBPS molecules hence lead the experimentally measured fluorescence increase. For highly abundant proteins, DMC treatment lead to fluorescence reduction in 1-D and 2-D gels for protein amounts higher than 250 ng. As higher protein amounts may bind more DMC molecules; the observed fluorescence attenuation was the result of enhanced RuBPS concentration, which might lead to concentration quenching.
The second additive process tested was the ultrasonication of entire polyacrylamide gel matrices containing RuBPS pre-stained proteins. Gel sonication might enable a destaining process by removing unbound RuBPS from the matrix. A significant reduction of background was found in 1-D gels and 2-D gels together with an increase of signal intensity. The results suggested that protein contents of 32 ng and higher were destainable by sonication to a threshold of about 4200 LAU. As the background fluorescence was uniformly destained, the result was a negative relative signal reduction that became measurable as net signal amplification for protein loads up to 259 ng.
It was concluded that sonication of polyacrylamide matrices was an effective way to reduce the background after RuBPS staining. Net signal amplification has the advantage of providing a better detection of low abundant proteins. However, background reduction should be done after protein quantification as the procedure has an impact on the dynamic the linearity of protein staining.
In conclusion, the presented experiments demonstrated that the tenside 1,3-Didecyl-2-methyl-imidazolium chloride enhanced the sensitivity of RuBPS staining to a comparable level as silverstaining for low abundant proteins. As DMC treatment was applied sequential to RuBPS staining precedent protein quantitation was possible. Therefore, DMC treatment could be a valuable additive to RuBPS staining for low abundant proteins that hitherto escaped detection. On the other hand, 1,3-Didecyl-2-methyl-imidazolium chloride quenched the fluorescence of high abundant proteins. Sonication of RuBPS stained gels lead to reduction of the background and a negative relative signal reduction which amplified the net signal for proteins loads up to 250 ng.
This study was done with the aim to impact the staining intensity of RuBPS what is in fact possible. By using DMC or sonication a very good staining technique is no longer quantifiable. The problem to make these procedures quantifiable is currently not solved. Nevertheless, the additional steps might be useful for researchers interested in visualization of low abundant proteins. As these data are not “publishable” via the “usual channels”, the authors decided to put this study on this webpage with the intention to provide the interested researcher the hint to do further experiments. Maybe some new helpful procedure will emerge from this. With the hope that somebody will find this useful the authors provide the results of this study for free download. Any correspondence about this topic is welcome and can be sent to firstname.lastname@example.org.
Prof. W. Bannwarth Albert Ludwig University, Freiburg i.B., Germany), the father of the RuBPS molecule, is acknowledged for the interesting discussions we had about RuBPS. Prof. Dr. André Haeberli, Department of Clinical Research, University of Berne, is acknowledged for providing the human blood serum samples.. Alain Zahn from the Department of Chemistry and Biochemistry, University of Bern for his literature search concerning DMC and Dr. Melek Turgut, Department of Pediatric Dentistry, Faculty of Dentistry, Hacettepe University, Ankara, Turkey, for the help with the manuscript.
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