Supporting Information for Proteomics DOI 10.1002/Pmic.200500596

Supporting Information for Proteomics DOI 10.1002/pmic.200500596 Li-Tai Jin, Sun-Young Hwang, Gyurng-Soo Yoo and Jung-Kap Choi A mass spectrometry compatible silver staining method for protein incorporating a new silver sensitizer in sodium dodecyl sulfate-polyacrylamide electrophoresis gels ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Supplementary Information A mass spectrometry compatible silver staining method for protein incorporating a new silver sensitizer in sodium dodecyl sulfate- polyacrylamide electrophoresis gels Li-Tai Jin, Sun-Young Hwang, Gyurng-Soo Yoo and Jung-Kap Choi College of Pharmacy, Chonnam National University, Kwangju 500-757, South Korea Correspondence: Prof. Jung-Kap Choi, Lab. of Analytical Biochemistry, College of Pharmacy, Chonnam National University, Yongbong-Dong 300, Buk- Ku, Kwangju 500-757, South Korea (Phone: +8262-530-2930; Fax: +8262-530- 2911; E-mail: [email protected]) Abbreviations used: EBT, eriochrome black T Keywords: Electrophoresis / Eriochrome black T / Mass spectrometry / Protein silver staining 1 1 Supplementary Introduction In life sciences, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a reliable and widely used analytical technique for the separation and identification of proteins. Given the development of proteomics, the ability to analyze and identify protein in a gel matrix at high sensitivity is becoming more important [1,2]. Various protein staining methods have been developed based on visible organic dyes, fluorescent and silver based systems, and radiolabeling. Of these, organic dyes are easily used but have low sensitivities, with detection limits in the range of 1-50 ng for proteins [2,3]. Fluorescent staining methods are a little more sensitive than dye-based methods but for data reading, special instruments are necessary that involve UV. These methods have detection limits that fall in the range of 0.1-10 ng for proteins [2,4]. Radiolabeling of proteins with radioactive isotopes is likely to remain the most sensitive method available. However, they are hazardous and require complicated handling procedures. With the possible exception of fluorescent staining and radiolabeling, silver staining offers greatest sensitivity for protein detection. Numerous silver staining protocols with different merits have been described [2,5,6,7,8]. However, relatively few are compatible with MS analysis. In the present study, we developed an eriochrome black T (EBT)-silver staining method that can detect 0.05-0.2 ng protein within 60 min while glutaraldehyde-silver method can only detect 0.2-0.6 ng protein within 100 min. The EBT dye (Fig. 1), which acts as a silver sensitizer, easily binds to 2 proteins and silver ions, and contains a diazo group, which reduces silver ions by cleavage in alkaline solution. We found that these properties of EBT significantly improve traditional silver staining in terms of sensitivity, speed, and MS compatibility. And more, the dye is cheap and widely used in other fields. So we believe that the developed EBT-silver method will be found useful for routine proteomics research. 2 Supplementary Materials and methods 2.1 Materials Acrylamide, Bis, TEMED, ammonium persulfate (APS), Tris base, SDS, eriochrome black T (EBT, dye content approx. 60%), iodoacetamide, glycerol, bromophenol blue, silver nitrate, sodium thiosulfate, potassium ferricyanide, formaldehyde, trypsin, potassium carbonate and molecular weight marker proteins (SDS-6H) including myosin heavy chain, β-galactosidase, phosphorylase b, bovine serum albumin (BSA), ovalbumin (OVA), carbonic anhydrase (CA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). EDTA, CHAPS, dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), urea, IPG strip, cover oil and IPG buffer were from Amersham Biosciences (Uppsala, Sweden). All other chemicals used were of analytical grade and were obtained 3 from various commercial sources. 2.2 Electrophoresis 2.2.1 Preparation for 1-DE Escherichia coli BL 21 (E. coli) was chosen for one-dimensional electrophoresis (1-DE) analysis. Total cell protein samples were obtained from E. coli using the following method. After cell culture, cells were harvested by centrifugation at 3,000 rpm for 10 min, sonicated in buffer containing 50 mM Tris, pH 7.5, 1 mM EDTA, and 0.4 mM PMSF for 5×1 min (1 g cell/10 ml buffer) and then centrifuged at 15,000 rpm for 20 min at 4. Protein concentrations in supernatant were ca. 16 mg/ml as determined by Bradford's method using a Bio-Rad protein assay kit. Molecular weight marker proteins (SDS-6H) and E. coli extract were dissolved in buffer containing 60 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 1% DTT, and 0.002% bromophenol blue for 1-DE. Prior to electrophoresis, protein samples were heated at 100 for 5 min in a boiling water bath and then cooled to room temperature. Twofold serial dilutions of marker proteins and E. coli samples were loaded onto gels at loadings of 100 ng to 0.2 ng/band and 4,000 ng to 8 ng/band, respectively. 4 Electrophoresis was carried out using polyacrylamide slab gels (60×80×0.75 mm) and a discontinuous buffer system [9]. 4.5% of stacking gel was overlaid on a 11.5% polyacrylamide separating gel with an acrylamide:Bis ratio of 30:0.8. The running buffer consisted of 0.025 M Tris, 0.2 M glycine, and 0.1% SDS. Gels were run in a Mini-protein III dual slab cell (Bio-Rad, Hercules, CA, USA) at a constant current of 22 mA per slab gel using a POWER PAC 300 supply (Bio- Rad). 2.2.2 Preparation for 2-DE The total cell protein samples of E. coli were separated using IPG gel strips (linear 4-7 pH gradient, 13 cm) for 2-DE analysis. Each strip loaded with 48 µg of sample was rehydrated in an Immobiline DryStrip Reswelling tray (Amersham Pharmacia Biotech, USA). Electrophoresis in the first dimension (isoelectric focusing) was performed using a Multiphor Electrophoresis unit (Amersham Pharmacia Biotech, USA) according to the manufacturer’s instructions. IPG strips containing the samples were transferred to the focusing tray and covered with mineral oil. Isoelectric focusing was performed using an EPS 3501 XL power supply (Amersham Pharmacia Biotech, USA) with a 5-step program (0 to 150 V for 1 h with gradient, 150 V to 300 V for 3 h, 300 V to 1,500 V for 1.5 h, 1,500 V to 3,500 V for 1.5 h, and hold at 3,500 V for 5 h). After isoelectric focusing, strips were removed and excess mineral oil was allowed drain off. Subsequently, strips 5 were incubated in 16 ml of first equilibration buffer (1% DTT, 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) for 15 min. For the second equilibration, the strips were incubated in 16 ml of second equilibration buffer (4% iodoacetamide, 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) for 15 min. The strips were placed in individual tubes with the support film toward the tube wall and the tubes were shaken on an adjustable tilt rocker (Labnet International Inc., Woodbridge, NJ, USA) during equilibration. The strips were transferred for electrophoresis in the second dimension. A separating gel of 11.5% polyacrylamide with an acrylamide:Bis ratio of 30:0.8 was cast using Hoefer SE 600 electrophoresis units (Amersham Pharmacia Biotech, USA). Gels were run at a constant current of 25 mA per slab in running buffer consisting of 0.025 M Tris, 0.2 M glycine, and 0.1% SDS. 2.3 Protein staining methods For all stainings, working solutions were prepared fresh with deionized water and clean glassware or plastic ware. All steps were carried out at room temperature with shaking. 2.3.1 EBT-silver staining 6 The EBT-silver staining procedure is summarized in Table 1. After electrophoresis, gels (0.75 or 1 mm thickness, 8×10 cm) were fixed in 200 ml of fixing solution (40 % v/v ethanol, 10% v/v acetic acid solution) for 2×20 min (or more to overnight). Gels were sensitized (stained) with 100 ml of staining solution containing 0.006% w/v EBT, 30% v/v ethanol solution by shaking for 2 min, and then destained using 200 ml of 30% ethanol solution for 2 min, and washed in 200 ml of deionized water for 2×2 min. Gels were then impregnated in 100 ml of 0.25% w/v silver nitrate containing 0.037% w/v formaldehyde for 5 min, washed in 200 ml of deionized water for 2×20 sec, and immersed in 100 ml of 2% w/v potassium carbonate, 0.04% w/v sodium hydroxide, 0.007% w/v formaldehyde, and 0.002% w/v sodium thiosulfate solution to develop image. After silver ion reduction, gels were immersed in 100 ml of 1.5% w/v EDTA for 10 min (or more to overnight) to stop the development. Stained gels were stored in deionized water or dried on filter paper under vacuum at 65 for 40 min. The EBT stock solution can be prepared 0.5% w/v with deionized water and stored for 1 year in dark. The volumes of the working solutions used in each step were proportional to the gel area, e.g., 400 ml of fixing solution for a 15×15 cm regular gel. 2.3.2 Glutaraldehyde-silver staining 7 Silver nitrate staining using glutaraldehyde as a sensitizer was performed using a modification of the method described by Heukeshoven and Dernick [8]. Briefly, after electrophoresis, gels (0.75 or 1 mm thickness, 8×10 cm) were fixed in 125 ml of 40% v/v ethanol, 10% v/v acetic acid solution for 30 min, and then reacted in 125 ml of 6.8% sodium acetate, 0.125% glutaraldehyde and 0.2% sodium thiosulfate solution for 30 min, and finally washed in 125 ml deionized water for 3×5 min.

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