1 of 5 8/21/2008 10:50 AM Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4233 Protocol Fluorescent Difference Gel Electrophoresis Angelika Görg, Oliver Drews, and Walter Weiss This protocol was adapted from "Fluorescent Difference Gel Electrophoresis," contributed by Angelika Görg, Oliver Drews, and Walter Weiss, Chapter 16, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004. INTRODUCTION A bottleneck for high-throughput proteomic studies is image analysis. In conventional two-dimensional (2D) methodology, protein samples are separated on individual gels, stained, and quantified, followed by image comparison with computer-aided image analysis programs. Since different images are not always perfectly superimposable, image analysis is very time consuming. Fluorescent difference gel electrophoresis shortens this procedure (Ünlü et al. 1997) by independently labeling samples in vitro before IEF using two different fluorescent cyanine dyes, then mixing and separating the samples on a single 2D gel. Subsequent image analysis enables the differences (e.g., up- or down-regulated proteins) between the samples to be visualized. MATERIALS Reagents Cell or tissue sample for protein extraction CyDye DIGE Fluor, Cy2, Cy3, Cy5 minimal dye (Amersham Biosciences) Dimethylformamide (DMF) (99.8% anhydrous) IMPORTANT: The DMF should be fresh (<3 months old after first opening), because it degrades to amine compounds, which interfere with the labeling reaction. Dithiothreitol (DTT) solution [50% (w/v)] Prepare fresh before use! 2DQuant kit (Amersham Biosciences) ph indicator strips (ph 4.5-10.0) Pharmalyte (ph 3-10) (Amersham Biosciences)
2 of 5 8/21/2008 10:50 AM SDS sample solubilization buffer Lysine (10 mm) Thiourea/urea/CHAPS buffer Equipment Image analysis software (e.g., DeCyder Differential Analysis Software; Amersham Biosciences) Low-fluorescence glass plate (3 mm thick) Multicolor confocal laser scanner (e.g., Typhoon 9400) Software for image overlay and editing (e.g., ImageQuant Solution [included in Typhoon package]) METHOD 1. Extract proteins with SDS sample solubilization buffer (see Extraction and Solubilization of Total Protein from Microorganisms). Determine the protein concentration of the sample with the 2DQuant kit. Dilute the SDS extract with a fourfold excess of thiourea/urea/chaps buffer to a protein concentration of 1 µg/µl and 20 µg/µl (final SDS concentration must be 0.2%). Adjust the ph of the sample to 8.5 (±0.1). IMPORTANT: Primary amines such as carrier ampholytes, as well as reducing agents such as dithiothreitol (DTT), diminish the labeling efficiency. Therefore, the common urea lysis buffer cannot be used for protein extraction. For a comprehensive list of partially or completely incompatible compounds, see the CyDye manual (from Amersham Biosciences). Proteins with low cystine content can be solubilized directly in thiourea/urea/chaps buffer. 2. Reconstitute the CyDyes in DMF to a stock solution of 1 mm. See the CyDyes manual for a detailed protocol. 3. Directly before labeling, dilute the CyDye stock solution to a final concentration of 0.4 mm (400 pmol/µl); for example, mix 3 µl of DMF with 2 µl of reconstituted CyDye. 4. Prepare an aliquot of 50 µg of the protein sample, and add 400 pmol (1 µl) of the CyDye. Keep the dyes and the samples in the dark and on ice! 5. Vortex the sample briefly, and centrifuge the mixture in a microfuge at 10,000g for 5 seconds. 6. Incubate the sample in the dark for 30 minutes on ice. 7. Add 1 µl of 10 mm lysine to stop the labeling reaction. 8. Mix, centrifuge briefly, and incubate in the dark for 10 minutes on ice.
3 of 5 8/21/2008 10:50 AM 9. Add 1 µl of DTT solution and 1 µl of Pharmalyte per 50 µl of sample solution. The final buffer concentration before isoelectric focusing (IEF) should not exceed 30 mm Tris. 10. Pool the protein samples that are going to be separated on the same first- and second-dimension gel, and immediately apply the mixture to the immobilized ph gradient (IPG) strip. Run IEF and SDS-PAGE as described in Isoelectric Focusing in Immobilized ph Gradient Strips Using the IPGphor Unit: Sample In-Gel Rehydration, Isoelectric Focusing in Immobilized ph Gradient Strips Using the IPGphor Unit: Sample Cup Loading, and Multiple SDS-PAGE on Vertical Electrophoresis Units. Approximately the same amount of fluorescence-labeled proteins should be loaded as would be needed for silver staining. Alternatively, store labeled sample for up to 3 months at 80ºC. Protect the IPGphor during the IEF and the buffer tank during SDS-PAGE from light to minimize photobleaching of the fluorescence dyes. 11. Following 2D gel electrophoresis, visualize the proteins using low-fluorescence glass plates. Directly scan the gels in the glass cassettes after the second dimension to ensure that all gels have the same dimensions; doing so simplifies spot matching of different gels. The exterior of the glass plates must be carefully cleaned with H 2 O and dried with a lint-free laboratory wipe before the gel cassette is positioned on the scanner. Each fluorescent dye should be consecutively excited to avoid fluorescence cross-talk and scanned at a resolution of at least 200 µm with a proper filter. After the scan, software such as ImageQuant Solution easily provides an initial image overlay of the scanned channels of one gel, thus giving a quick overview of differences between the labeled extracts. It should be kept in mind that the protein patterns of one gel might differ slightly in protein concentration and background. Although the DIGE system is highly reliable in terms of quantitation, especially when an internal standard is included in the experimental design, biological variances or variances during the protein extraction might affect samples. Therefore, at least three gels of three individual protein extracts per experimental condition (e.g., control and stress) should be run, normalized, and compared before qualitative and quantitative changes can be considered significant. REFERENCES Ünlü M., Morgan M.E., Minden J.S. 1997. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 18: 2071 2077.[Medline] Caution Dimethylformamide (DMF) Dimethylformamide (DMF; also known as N,N-dimethylformamide) HCON(CH 3 ) 2 is irritating to the eyes, skin, and mucous membranes. It can exert its toxic effects through inhalation, ingestion, or skin absorption. Chronic inhalation can cause liver and kidney damage. Wear appropriate gloves and safety glasses. Use in a chemical fume hood.
4 of 5 8/21/2008 10:50 AM Caution Dithiothreitol (DTT) Dithiothreitol (DTT) is a strong reducing agent that emits a foul odor. It may be harmful by inhalation, ingestion, or skin absorption. When working with the solid form or highly concentrated stocks, wear appropriate gloves and safety glasses and use in a chemical fume hood. Recipe SDS sample solubilization buffer 1% (w/v) SDS 100 mm Tris-HCl (ph 9.5) To prepare 50 ml of SDS sample buffer, dissolve 0.5 g of SDS and 0.6 g of Tris base in ~40 ml of deionized H 2 O. Titrate to ph 9.5 with 4 N HCl, filter, and adjust the volume to 50 ml with deionized H 2 O. Recipe Thiourea/urea/CHAPs buffer 2 M thiourea 7 M urea 4% (w/v) CHAPS To prepare 50 ml of buffer, dissolve 22.0 g of urea and 8.0 g of thiourea in ~25 ml of deionized H 2 O. Add 0.5 g of Serdolit MB-1 ion-exchange resin (Serva), stir for 10 minutes, and filter. Add 2.0 g of CHAPS to the filtrate and adjust the final volume to 50 ml with deionized H 2 O. Copyright 2006 by Cold Spring Harbor Laboratory Press. Online ISSN: 1559-6095 Terms of Service All rights reserved. Anyone using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used with caution. For a full listing of cautions, click here. All rights reserved. No part of these pages, either text or images, may be used for any reason other than personal use. Reproduction, modification, storage in a retrieval system or retransmission, in any form or by any means-electronic, mechanical, or otherwise-for reasons other than personal use is strictly prohibited without prior written permission.
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