Proteome © Springer-Verlag 2000 DOI 10.1007/s102160000002
Original Article
Ruthenium II tris (bathophenanthroline disulfonate), a powerful fluorescent stain for detection of proteins in gel with minimal interference in subsequent mass spectrometry analysis Thierry Rabilloud 1, , Jean-Marc Strub 2 , Sylvie Luche 1 , Jean Luc Girardet 3 , Alain van 2 Dorsselaer and Joël Lunardi 1 (1)
CEA-Laboratoire de Bioénergétique Cellulaire et Pathologique, EA 2943, DBMS/BECP, CEA-Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9, France
(2)
Laboratoire de Spectrométrie de Masse Bio-Organique, UMR CNRS 7509, 1, rue Blaise Pascal, 67008 Strasbourg Cedex, France
(3)
CEA - Laboratoire de Biophysique Moléculaire et Cellulaire, UMR CNRS 5090, DBMS/BMC, CEA-Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9, France E-mail:
[email protected] Phone: +33-76-883212 Fax: +33-76-885187
Received: 1 June 2000 / Accepted: 20 July 2000 / Published online: 31 August 2000 Abstract. Fluorescent ruthenium chelates have been prepared and investigated for staining proteins separated by electrophoresis. Ruthenium II tris (bathophenanthroline disulfonate) appears to be suitable for detection of proteins, with sensitivities in the nanogram range, although sensitivity does not reach the level of optimized silver staining methods. This compound can be efficiently detected either with UV tables or with commercial laser fluorescence scanners. A further advantage of this detection method is its compatibility with mass spectrometry., These factors make this method a good compromise for proteomics studies.
Introduction Fluorescent detection of proteins after gel electrophoresis is a technique that has recently re-emerged. While fluorescent labelling of proteins is very widely used in immunological detections, this has not been the case in general protein detection coupled to electrophoretic separations. The oldest fluorescent detection schemes involve labelling of proteins prior to electrophoresis, generally with amine-reactive probes such as FITC (Duffy et al. 1989), then separation of proteins by electrophoresis
-1-
prior to final detection with a film, a CCD camera or a fluorescence scanner. Other probes described in this scheme are cysteine-reactive probes (Kirley 1989), which offer a similar level of detection but fewer coupling artefacts. This pre-electrophoresis covalent coupling scheme has never reached widespread use because of several associated artefacts. For example, in the case of 2D electrophoresis, pre-electrophoresis labelling is complicated by the fact that the pI should not be modified. Pre-labelling has therefore been carried out either with neutral, thiol-reactive probes (Urwin and Jackson 1993) or with amino-reactive probes bearing a single positive charge in order to replace the protein charge lost by coupling the probe (Unlü et al. 1997). However, problems will be encountered when basic proteins are analysed. In these cases, the real pKas of thiol and amino groups must be respected in order to have access to the true pI, and this is not the case with the probes used to date for pre-labelling. Even for neutral and acidic proteins, artefacts in the apparent molecular weight are quite common (Unlü et al. 1997). Consequently, the position of the fluorescent spot will be slightly offset with respect to the position of the bulk of the protein, leading to problems when the spot must be excised for subsequent analysis. Solubility decreases induced by the grafting of the fluorescent group are also quite common (Unlü et al. 1997). Other covalent probes, irrespective of the pI of the proteins, can also be used, especially in SDS polyacrylamide gel electrophoresis (PAGE). In 2D electrophoresis, they must be grafted either between IEF and SDS-PAGE (Urwin and Jackson 1991) or after electrophoresis. In the latter case, molecules which are non-fluorescent but become fluorescent after coupling are generally chosen, such as fluorescamine (Jackowski and Liew 1980), or 2-methoxy-2,4-diphenyl furanone (MDPF) (Jackson et al. 1988). This is due to the fact that any fluorescent molecule remaining in the gel will induce a very high background. However, all these covalent probes have the general drawback of chemically modifying the protein, which strongly hampers subsequent protein analysis by Edman sequencing or mass spectrometry. This is especially true for amine-reactive probes, because numerous amine sites exist on proteins. Cysteine-reactive probes are much less prone to this drawback, as cysteines are much less frequent then lysines in proteins. However, cysteine is very unevenly distributed in proteins. This implies in turn than cysteine-rich proteins (e.g. albumin, thioredoxins, metallothioneins) will be grossly overlabelled, while many other proteins (e.g. carbonic anhydrase) do not contain a single cysteine and will therefore remain completely undetected. Such cysteine-lacking proteins are not uncommon, as they represent 14% of all currently available Swiss-Prot entries. However, the enormous intrinsic advantages of fluorescent detection, i.e. sensitivity down to a few attomoles of probes, as shown in DNA sequencing (Smith et al. 1986), and linearity over several orders of magnitude (up to five), have prompted continuous research in this field to overcome the limitations of covalent labelling. The most frequently used method is to use non-covalent, environment-sensitive probes. Generally, these probes are very weakly fluorescent in water but highly fluorescent in apolar media, such as detergent. Advantage is then taken of the binding of detergent to proteins to build a fluorescence-promoting environment at the protein sites in the gel. One of the best-known probes of this type is ANS (anilino naphthalene sulfonate). Its application to fluorescent detection of proteins in gels was described some years ago, with a sensitivity close to that of dye detection (Daban and Aragay 1984). The use of more sensitive probes, such as BisANS (Horowitz and Bowman 1987), and more recently Sypro dyes from Molecular Probes (Steinberg et al. 1996), or Nile Red (Alba et al. 1996) has considerably improved the sensitivity of this approach. This non-covalent method has, however, a limited contrast by definition. Ideally detergent should be present on the proteins, but no apolar environment (i.e. no micelles) should be present in the gel. This is almost impossible to achieve, as any treatment disrupting the micelles (and thus reducing the background) will also decrease the
-2-
amount of detergent bound to the proteins (and thus the signal). The commonly suggested lowering of SDS concentration in the gel to avoid the presence of micelles (e.g. as in Alba et al. 1996) is acceptable for 1D electrophoresis but often leads to vertical streaking when applied to 2D electrophoresis. This limited contrast lowers the sensitivity, which approaches the sensitivity of silver staining only with extensive exposure on a light integrating detection system (film, CCD camera). In scanning devices, the sensitivity is closer to that of colloidal Coomassie blue (Neuhoff et al. 1988), i.e. one order of magnitude worse than silver (Rabilloud and Charmont 1999). However, as expected from non-covalent, post-electrophoresis labelling, there are neither migration artefacts linked to coupling of a probe, nor problems linked to modification of the proteins in subsequent analyses. Thus, another principle is desirable for fluorescent detection of proteins after gel electrophoresis. This staining method should be non-covalent but should offer more contrast than the SDS micelles-based methods. The principle of Coomassie blue staining, i.e. staining without destaining with a very low concentration of free dye, offers an interesting choice. The best choice would be to have a colloidal system (Neuhoff et al. 1998), but this is often not possible. Alternatively, it is possible to use very low concentrations but large volumes of the staining agent (Chen et al. 1993). Although time-consuming, this method offers good contrast and reproducible results. When transposed to fluorescence, this corresponds to using a fluorescent compound which binds non-covalently to proteins. By analogy with classical protein dyes, chemicals containing multiple sulfonic acid groups are the compounds of choice. The examples include many organic dyes (Wilson 1983), but also metal-organic chelates (Graham et al. 1978). In addition, the fluorescent probe should be excitable both in UV and visible light, if possible close to the emission bands of classical lasers, and also light-resistant to prevent fading. Sulfo derivatives of classical organic fluorophores (e.g. sulfo rhodamines) do not bind with sufficient avidity to proteins to give useful staining methods. However, metal chelates made with the chelator bathophenanthroline disulfonate bind strongly to proteins (Graham et al. 1978). While the iron chelate described in this paper is not fluorescent, a fluorescent europium chelate has been described with very good sensitivity (Lim et al. 1997). However, this europium chelate is excited only with UV light. This limits detection to setups with film (i.e. non-linear) or with CCD cameras, which do not offer the resolution (0.1 mm) required for the analysis of 2D gels and/or are prone to saturation effects. Therefore, a laser-excitable chelate would be desirable. In this area, ruthenium II-phenanthroline chelates have been described for DNA detection (Bannwarth 1989) and seem to have the required features. We therefore decided to prepare the ruthenium II-bathophenanthroline disulfonate chelate, i.e. the ruthenium version of the chelates previously shown to be applicable to protein detection (Graham et al. 1978; Lim et al. 1997), and to test it for detection of proteins after electrophoresis.
Materials and methods Fluorescent probe synthesis Ruthenium II-bathophenanthroline disulfonate chelate was prepared essentially as described previously (Lin et al. 1976), with slight modifications: 0.5 g of potassium pentachloro aquo ruthenate (K 2 Cl 5 Ru.H 2 O), purchased from Alfa Aesar (26.9% Ru) was dissolved in 50 ml boiling water and kept under reflux, resulting in a deep red-brown solution. Three molar equivalents of bathophenanthroline disulfonate, disodium salt (Alfa), i.e. 2.2 g of the anhydrous compound, were added and the refluxing continued for 20 min. The solution turned to a deep greenish brown. Meanwhile, a reducing solution containing 12 mM of sodium hydroxide and 8 mM of phosphinic acid (Fluka) in 10 ml water was prepared. This solution was then added to the refluxing mixture and refluxing was continued for another 20 min. The solution turned to a deep orange-brown. After cooling, the pH was adjusted to 7 with sodium hydroxide and the volume was adjusted to 65 ml with
-3-
water. This gives a 20 mM stock solution, which can be stored in the fridge for several months. The absorption spectra were recorded on a JASCO-V-530 spectrophotometer. The fluorescence spectra (excitation and emission) of the chelate were recorded on a SPEX Fluoromax spectrofluorometer. Alternatively, the reducing solution could be prepared with 8 mM of sodium ascorbate dissolved in 10 ml of water.
Gel electrophoresis Proteins were separated by SDS-PAGE, either in the standard tris-glycine system, or in the tris-taurine system (Rabilloud et al. 1994a). For 2D separation, the first dimension was an isoelectric focusing with immobilized pH gradients and sample application by in-gel rehydration (Rabilloud et al. 1994a), using a urea-thiourea mixture as solubilizing agent (Rabilloud et al. 1997).
Detection of proteins after electrophoresis Proteins were detected after electrophoresis either with colloidal Coomassie blue (Neuhoff et al. 1988), Sypro orange (Steinberg et al. 1996) or silver (Rabilloud et al. 1994b). They were otherwise detected with the ruthenium chelate as follows: following electrophoresis, gels were thoroughly fixed (at least four times for 30 min, but preferably overnight) in 30% (v/v) ethanol-10% (v/v) acetic acid. The gels were then rinsed four times for 30 min in 20% ethanol. Thorough removal of acetic acid is required, as acids strongly quench the fluorescence of the chelate. The gels were then stained for 3 h-6 h in 20% ethanol containing 100-200 nM of ruthenium chelate, i.e. 5 µl-10 µl of stock solution per litre of staining solution. Finally, the gels were reequilibrated in water (twice for 10 min) prior to imaging, either on a 302 nm UV table or with a laser scanner equipped with a 488 nm or 532 nm laser. We used a Molecular Dynamics Fluorimager with a 488 nm laser.
Mass spectrometry analysis Stained protein spots or bands were excised (on a UV table for fluorescent detection), and destained in 50% ethanol. Silver-stained proteins were destained with ferricyanide-thiosulfate (Gharahdaghi et al. 1999), and the gel finally shrunk in 50% ethanol. Each gel slice was cut into small pieces with a scalpel, washed with 100 µl of 25 mM NH 4 HCO 3 , and agitated for 8 min with a Vortex mixer. After settling of the gel pieces, the supernatant was removed. Gel pieces were dehydrated with 100 µl of acetonitrile, agitated for 8 min with a Vortex mixer. After settling of the gel pieces, the supernatant was removed. This operation was repeated twice. Gel pieces were completely dried with a Speed Vac (15 min) before reduction-alkylation. Gel pieces were covered with 100 µl of 10 mM DTT in 25 mM NH 4 HCO 3 and the reaction was left to proceed at 57°C for 1 h. The supernatant was removed, 100 µl of 55 mM iodoacetamide in 25 mM NH 4 HCO 3 were added and the reaction was left in the dark at room temperature for 1 h. The supernatant was removed and the washing procedure with 100 µl of NH 4 HCO 3 and acetonitrile was repeated three times. Gel pieces were completely dried with a Speed Vac (15 min) before tryptic digestion. The dried gel volume was evaluated and three volumes of trypsin (12.5 ng/µl) in 25 mM NH4HCO3 (freshly diluted) were added. The digestion was performed at 35°C overnight. The gel pieces were centrifuged and 5 µl of 25% H 2 O/70% acetonitrile/5% HCOOH were added. The mixture was sonicated for 5 min and centrifuged. The supernatant was recovered and the operation was repeated once. The supernatant volume was reduced under nitrogen flow to 4 µl, 1 µl of H 2 O/5% HCOOH were added and 0.5 µl of the mix were used for MALDI-TOF analysis.
-4-
Mass measurement were carried out on a Brucker BIFLEX matrix-assisted laser desorption time-of-flight mass spectrometer (MALD-TOF) equipped with the SCOUT High Resolution Optics with X-Y multisample probe and gridless reflector. This instrument was used at a maximum accelerating potential of 20 kV and was operated in reflector mode. Ionization was carried out with a 337 nm beam from a nitrogen laser with a repetition rate of 3 Hz. The output signal from the detector was digitized at a sampling rate of 1 GHz. A saturated solution of -cyano-4-hydroxycinnamic acid in acetone was used as a matrix. A first layer of fine matrix crystals was obtained by spreading and fast evaporation of 0.5 µl of matrix solution. On this fine layer of crystals, a droplet of 0.5 µl of aqueous HCOOH (5%) solution was deposited. Afterwards, 0.5 µl of sample solution was added and a second 0.2 µl droplet of saturated matrix solution (in 50% H 2 O/50% acetonitrile) was added. The preparation was dried under vacuum. The sample was washed one to three times by applying 1 µl of aqueous HCOOH (5%) solution on the target and then flushed after a few seconds. Calibration was performed in internal mode with four peptides, angiotensin (1046.542 Da), substance P (1347.736 Da), bombesin (1620.807 Da), and ACTH (2465.199 Da).
Results and discussion Chelate characterization Two ruthenium chelates were prepared initially, the tris-bathophenanthroline disulfonate chelate (RuBPS) and the tris ferrozine chelate (see Fig. 1). Both organic moieties bear a bipyridine group, which seems essential to obtain fluorescent chelates (Lin et al. 1976). However, only the bathophenanthroline chelate proved fluorescent. This might be due either to an intrinsic property of the ferrozine chelate, or to the fact that ruthenium could not be reduced by our reducing mixes in the ferrozine chelates. It must be stressed that only Ru II, and not Ru III, chelates are fluorescent. In the preparation method described above, a Ru III chelate was first prepared by mixing the Ru salt and the organic chelator. This chelate was then reduced in situ to a Ru II chelate, in our case by phosphinic acid or ascorbic acid. Other reducing agents such as molecular hydrogen have also been described (Lin et al. 1976). When reduction is successful, the resulting solution gives an intense pink fluorescence when diluted 1/1,000 in water and exposed to UV (302 nm) light. The absorption and emission spectra are shown in Fig. 2. There are two absorption bands, a narrow band around 276 nm and a broad one in the 425 nm-500 nm range. The fluorescence emission spectra exhibit a single maximum at around 601 nm, both for UV and for visible excitation. The excitation spectrum for a 600 nm emission showed broad excitation bands in the 300 nm range and in the 400 nm-500 nm range (not shown).
-5-
Fig. 1A-C. Structure of ruthenium complexes. A Three-dimensional structure of Ru II tris (bathophenanthroline disulfonate); B planar structure of Ru II tris (bathophenanthroline disulfonate); C planar structure of Ru II tris (ferrozine)
-6-
Fig. 2. Absorption and emission spectra of a 50 µM Ru II tris (bathophenanthroline disulfonate) solution in water It appeared, however, on repeated synthesis with various phosphinic derivatives that reduction could be erratic. In this case, the brown colour of the chelate persisted, but no fluorescence occurred. It was then observed that low pH was frequently correlated with such poor batches. It was shown that diluting a good chelate batch at low pH inhibited the fluorescence per se. Furthermore, reduction by phosphinic acid is also pH dependent, so that reduction at too low a pH followed by pH correction with NaOH did not give correct batches. For these reasons, reduction with sodium ascorbate was preferred, as it did not give rise to these pH-associated problems.
Staining with RuBPS chelate Several staining schemes were tested. In the first, the gel was stained with millimolar or submillimolar concentrations of chelate (Graham et al. 1978), followed by destaining. No contrast was observed in this case, the gel being either absolutely non-fluorescent (too strong destaining) or with an enormous background fluorescence (too weak destaining). We could not find conditions offering correct contrast. In a second set of experiments, we first determined the concentrations offering minimal background fluorescence. Concentrations of 100 nM-200 nM were chosen. In order to build a versatile stain offering the possibility of further protein analysis, we decided to use an acid-alcohol fixation. This fixation was also believed to convert the proteins to a cationic form, in which they could therefore bind avidly the anionic chelate. However, the pH sensitivity of the chelate necessitated thorough rinsing prior to staining. Rinses and stains contained 20% ethanol to limit protein diffusion in the gel. Long staining times (at least 3 h) were required for optimal detection. A slight increase in sensitivity was observed if the staining time was prolonged to 6 h. With an optimal staining scheme, sensitive detection of proteins could be achieved. The absolute sensitivity was first determined by staining serial dilutions of molecular weight markers separated by SDS-PAGE (Fig. 3a). This detection method was compared to colloidal Coomassie blue staining (Fig. 3b), Sypro orange (Fig. 3c) or silver staining (Fig. 3d). It appeared that RuBPS was at least two-fold more sensitive than Coomassie blue or Sypro orange, and two- to five-fold less sensitive than
-7-
silver.
-8-
Fig. 3A-D. Comparison of detection by fluorescence with silver staining and colloidal Coomassie blue. Serial dilutions of molecular weight markers (Bio-Rad, broad range) were separatedby SDS PAGE (1.5 mm thick gels, 10% acrylamide). The gel was then stained with RuBPS (A), colloidal Coomassie blue (B), Sypro orange (C), or silver (D). Amounts loaded on the gel lanes (in ng per band, from left to right): 400 ng, 200 ng, 100 ng, 50 ng, 20 ng, 10 ng, 5 ng Staining homogeneity over a wide range of proteins was explored using 2D electrophoresis (Fig. 4). Here again, RuBPS performed significantly better than Coomassie blue, but not as well as silver staining. It must be noted, however, that a decrease in sensitivity was observed when several 2D gels were stained overnight in the same container with 100 nM RuBPS. This was attributed to an insufficient amount of RuBPS under those conditions. It seems that the chelate first diffuses uniformly into the gel and binds to all the proteins present, thereby giving good detection. However, if the amount of RuBPS is insufficient and the staining time long enough, the high-abundance proteins act as sinks and displace the probe from the less abundant proteins, thereby decreasing the detection threshold. If overnight staining of multiple gels is to be achieved, the RuBPS concentration must be optimized first.
-9-
Fig. 4A-C. Comparison of detection by fluorescence with silver staining and colloidal Coomassie blue. A total extract prepared from Jurkat cells was separated by 2D gel electrophoresis. Two hundred micrograms were loaded on the first dimension gel (IPG (Immobilized pH Gradient), pH4-8). The gels were then stained with RuBPS (A), colloidal Coomassie blue (B), or silver (C). Second dimension gels: 10% acrylamide Linearity of the stain was not thoroughly evaluated. However, the peak height followed the amount of molecular weight proteins loaded on 1D gels, which was not the case for silver staining.
Interference with mass spectrometry In addition to poor linearity, poor interface with subsequent protein analysis techniques is a drawback of silver staining methods. Although several mass spectrometry-compatible methods have been described (Shevchenko et al. 1996; Rabilloud et al. 1998), the yield of peptides seems to be erratic, and in any case much lower than with dye staining (Gevaert and Vandekerckhove 2000). We therefore excised several equivalent spots on 2D gels stained with RuBPS, silver or Coomassie blue, and submitted them to MALDI-TOF analysis. Typical spectra are shown in Fig. 5. It appeared that RuBPS performed at least as well as Coomassie blue and significantly better than silver.
- 10 -
Fig. 5A-C. Comparison of mass spectrometry spectra obtained from the same 2D spot (hsp90). Identical samples (whole cell extracts from Jurkat cells) were loaded on 2D gels stained with RuBPS (A), colloidal Coomassie blue (B), or silver (C). Note the clearly lower peaks present in the silver-stained spot, even after destaining, compared to the Commassie or ruthenium-stained spots (e.g. peaks at 1194 to 1249, 1527, 1782 to 1847, 2015 Da)
Concluding remarks The harmonious combination of good sensitivity, good linearity and minimal interference with MALDI-TOF analysis make RuBPS fluorescent staining an attractive choice, especially for analysis of 2D gel-separated proteins. Moreover, the very low concentrations used make this stain very cost-effective. The only difficulty is the preparation of the chelate, which seemed to be erratic in some attempts. Due to these difficulties and to the low concentration needed, it is recommended that small batches (starting with 0.2 g K 2 RuCl 5 ) are prepared, thereby minimizing chemical wastage should a synthesis problem occur. While this work was in progress, another ruthenium-based fluorescent stain was cursorily described (Berggren et al. 1999; Steinberg et al. 2000) and commercially introduced (Sypro Ruby from Molecular Probes). The comparison of the excitation and emission spectra of Sypro Ruby and RuBPS suggests that Sypro Ruby may contain RuBPS. However, the differences in the optimal protocols suggest that Sypro Ruby is not a simple solution of RuBPS. While the various Sypro Ruby formulations offer ready-to-use, optimized formulas, the RuBPS protocol described here is much more cost-effective and can probably be optimized to other protein separation setups such as IEF gels or blots.
- 11 -
References Alba FJ, Bermudez A, Bartolome S, Daban JR (1996) Detection of five nanograms of protein by two-minute Nile red staining of unfixed SDS gels. Biotechniques 21:625-626 Bannwarth W (1989) Bathophenanthroline-Ru(II) complexes as nonradioactive labels for dideoxy DNA sequencing. Anal Biochem 181:216-219 Berggren K, Steinberg TH, Lauber WM, Carroll JA, Lopez MF, Chernokalskaya E, Zieske L, Diwu Z, Haugland RP, Patton WF (1999) A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports. Anal Biochem 276:129-143 Chen H, Cheng H, Bjerknes M (1993) One step Coomassie Brilliant Blue R250 staining of proteins in polyacrylamide gels. Anal Biochem 212:295-296 Daban JR, Aragay AM (1984) Rapid fluorescent staining of histones in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 138:223-228 Duffy VE, Stockwell PA, Warrington DM, Monk BC (1989) Laser excitation of fluorescent-labeled polypeptides in polyacrylamide gels. Anal Biochem 179:291-298 Gevaert K, Vandekerckhove J (2000) Protein identification methods in proteomics. Electrophoresis 21:1145-1154 Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20:601-605 Graham G, Nairn RS, Bates GW (1978) Polyacrylamide gel staining with Fe 2+ -bathophenanthrolinesulfonate. Anal Biochem 88:434-441 Horowitz PM, Bowman S (1987) Ion-enhanced fluorescence staining of sodium dodecyl sulfate-polyacrylamide gels using bis (8-p-anilino-1-naphthalenesulfonate). Anal Biochem 165:430-434 Jackowski G, Liew CC (1980) Fluorescamine staining of nonhistone chromatin proteins as revealed by two-dimensional polyacrylamide gel electrophoresis. Anal Biochem 102:321-325 Jackson P, Urwin V, Mackay CD (1988) Rapid imaging, using a cooled charge-coupled-device, of fluorescent two-dimensional polyacrylamide gels produced by labelling proteins in the first dimensional isoelectric focusing gel with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone. Electrophoresis 9:330-339 Kirley TL (1989) Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analyses. Anal Biochem 180:231-236 Lim MJ, Patton WF, Lopez MF, Spofford KH, Shojaee N, Shepro D (1997) A luminescent europium complex for the sensitive detection of proteins and nucleic acids immobilized on membrane supports. Anal Biochem 245:184-195
- 12 -
Lin CT, Böttcher W, Chou M, Creutz C, Sutin N (1976) J Am Chem Soc 98: 6536-6544 Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255-262 Rabilloud T, Charmont S (1999) Detection of proteins on two-dimensional electrophoresis gels. In: Rabilloud T (ed) Proteome research: two-dimensional gel electrophoresis and identification methods. Springer, Berlin Heidelberg New York, pp 107-126 Rabilloud T, Valette C, Lawrence JJ (1994a) Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 15:1552-1558 Rabilloud T, Vuillard L, Gilly C, Lawrence JJ (1994b) Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol 40:57-75 Rabilloud T, Adessi C, Giraudel A, Lunardi J (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18:307-316 Rabilloud T, Kieffer S, Procaccio V, Louwagie M, Courchesne PL, Patterson SD, Martinez P, Garin J, Lunardi J (1998) Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: toward a human mitochondrial proteome. Electrophoresis 19:1006-1014 Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850-858 Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB, Hood LE (1986) Fluorescence detection in automated DNA sequence analysis. Nature 321:674-679 Steinberg TH, Jones LJ, Haugland RP, Singer V (1996) SYPRO orange and SYPRO red protein gel stains: one-step fluorescent staining of denaturing gels for detection of nanogram levels of protein. Anal Biochem 239:223-237 Steinberg TH, Chernokalskaya E, Berggren K, Lopez MF, Diwu Z, Haugland RP, Patton WF (2000) Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain. Electrophoresis 21:486-496 Unlü M, Morgan EM, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071-2077 Urwin VE, Jackson P (1991) A multiple high-resolution mini two-dimensional polyacrylamide gel electrophoresis system: imaging two-dimensional gels using a cooled charge-coupled device after staining with silver or labelling with fluorophore. Anal Biochem 195:30-37 Urwin VE, Jackson P (1993) Two-dimensional polyacrylamide gel electrophoresis of proteins labelled with the fluorophore monobromobimane prior to first-dimensional isoelectric focusing: imaging of the fluorescent protein spot patterns using a cooled charge-coupled device. Anal Biochem 209:57-62
- 13 -
Wilson CM (1983) Staining of proteins on gels: comparisons of dyes and procedures. Methods Enzymol 91:236-247
- 14 -
