npgrj_nProt_234 1351..1358
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1. 643 656 2004 9 Shaw M M amp Riederer B M Sample preparation for two dimensional gel electrophoresis Proteomics 3 1408 1417 2003 10 Marouga R David S amp Hawkins E The development of the DIGE system 2D fluorescence difference gel analysis technology Anal Bioanal Chem 382 669 678 2005 1358 VOL 1 NO 3 2006 NATURE PROTOCOLS 11 12 13 14 15 16 17 18 19 20 21 22 Okano T et al Plasma proteomics of lung cancer by a linkage of multi dimensional liquid chromatography and two dimensional difference gel electrophoresis Proteomics 6 3938 3948 2006 Wang H et al Intact protein based high resolution three dimensional quantitative analysis system for proteome profiling of biological fluids Mol Cell Proteomics 4 618 625 2005 Kondo T et al Application of sensitive fluorescent dyes in linkage of laser microdissection and two dimensional gel electrophoresis as a cancer proteomic study tool Proteomics 3 1758 1766 2003 Sitek B et al Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling Proteomics 6 4506 4513 2006 Zhou G et al 2D differential in gel electrophoresis for the identification of esophageal scans cell cancer specific protein markers Mol Cell Proteomics 1 117 124 2002 Karp N A amp Lilley K S Maximising sensitivity for detecting changes in protein exp2. Scanalytics QuickTime SExtractor Protein digestion robot e g ProGest Genomics Solutions REAGENT SETUP A CRITICAL Wear gloves at all times to avoid keratin contamination which is a key obstacle to further analysis by MS Lysis buffer 7 M urea 2 M thiourea 4 wt vol CHAPS 10 mM DTT and 10 mM Na HEPES pH 8 0 A CRITICAL Never heat urea solutions above room temperature 18 21 C as this will cause the formation of cyanate which carbamylates protein and produces charge trains in 2DE gels The pH of this solution is critical for the labeling reaction We typically store the lysis buffer in 1 ml aliquots at 80 C Dye solutions Add 25 ul DMF to each 25 nmol tube of Cy3 NHS As the extinction coefficient of Cy5 is greater than that of Cy3 add 37 5 ul DMF to each 25 nmol tube of Cy5 NHS A CRITICAL Use fresh dry DMF of the best quality Store dye solutions at 80 C and allow them to equilibrate to ice temperature before opening the tube Quenching solution 5 M methylamine HCl and 10 mM HEPES pH 8 0 Dissolve 2 38 g HEPES in 38 8 ml of 40 methylamine aqueous solution Slowly add concentrated HCl with stirring until the pH reaches 8 0 1 CAUTION This step should be done on ice in a fume hood You will need 60 ml acid to reach the desired pH The total volume should be 100 ml Aliquot and store at 80 C Rehydration buffer 7 M urea 2 M thiourea 4 wt vol CHAPS 10 mM DTT 2 mM acetic acid 0 002 wt vol bromo
3. 6 12 h 13 IEF should be set up according to the manufacturer s instructions with a few modifications Refer to the user manual for more details and precautions to be taken while setting up IEF Turn on the IPGphor Place ceramic strip holders on the IPGphor platform with the pointed end towards the anode 14 Cut two 3 mm filter paper wicks for each gel and immerse in HPLC water The wicks should be approximately the width of the gel They can be made a little longer than they are wide 15 Open the rehydration cassette assembly Remove a rehydrated IEF strip and rinse it briefly with HPLC water Blot excess water Place the IEF strip in the holder with the acidic end towards the anode Place wicks at both ends of the strip making sure to blot excess water from the wicks before placing on the gel Place an electrode on each of the wicks Place a sample cup near the electrode on the acidic end of the gel Cover the surface of the gel with 1 ml of DryStrip Cover Fluid You can also cover the entire length of the gel between the cathodic electrode and the sample cup holder with nylon mesh strips cut to the width of the opening in the ceramic strip holder and the length of the exposed IEF strip Gently press down with tweezers to make sure the strips are contacting the surface of the gel The nylon mesh helps retain a thin film of cover fluid over the surface of the gel thereby preventing it from drying out A CRITICAL STEP Avoid unnecessary delays durin
4. BSA in lysis buffer to each tube as a loading control Mix by vortexing 10 Add 1 ul Cy3 NHS stock solution to tubes 1 and 4 Add 1 ul Cy5 NHS stock solution to tubes 2 and 3 Mix by vortexing Incubate in the dark on ice for 15 min To synchronize the labeling time add the dye to the walls of all reaction tubes The Cy3 and Cy5 reaction tubes containing samples to be loaded on the same gel are briefly spun vortexed and spun again in a microcentrifuge at top speed for a few seconds at 4 C A CRITICAL STEP Allow the dye stocks to warm up to ice temperature before opening 11 To each tube add 1 ul quenching solution Again the solution is dispensed onto the walls of the tubes and the reaction is started as described in Step 10 Incubate in the dark on ice for 30 min The duration of quenching can be extended up to 2 h if necessary PAUSE POINT After labeling the samples can be stored at 80 C Thaw on ice prior to applying to the IEF strips 12 In a new tube labeled gel 1 combine the entire contents of tubes 1 and 2 In a new tube labeled gel 2 combine the contents of tubes 3 and 4 Add 1 6 ul of the appropriate IPG buffer solution 1 ul for every 100 ul of sample to the gel 1 and 2 tubes Mix by vortexing and briefly spin in a microcentrifuge at top speed for a few seconds at 4 C to consolidate all of the liquid The samples should be loaded onto IEF strips immediately after combining First dimension IEF TIMING
5. and difficult to model Difference gel electrophoresis DIGE was developed to over come the irreproducibility problem in the 2DE methodology by labeling two samples each with a different fluorescent dye prior to running them on the same gel Fig 1 The fluorescent dyes used in DIGE Cy3 NHS and Cy5 NHS Fig 2 are cyanine based molecular weight matched amine reactive and positively charged These characteristics coupled with substoichiometric labeling result in no electrophoretic mobility shifts arising between the two differentially labeled samples when they are co electrophor esed Therefore in DIGE every identical protein in one sample superimposes with its differentially labeled counterpart in the other sample allowing for more reproducible and facile detection of differences Furthermore DIGE is a sensitive technique capable of detecting as little as 0 5 fmol of protein and this detection system is linear over a gt 10 000 fold concentration range gt The most important considerations in performing DIGE experi ments are experimental design and sample preparation DIGE has been used to analyze proteome changes from a wide variety of cell types and bodily fluids including serum The sample pre paration protocol depends on the cell type Most samples require mild homogenization in lysis buffer to extract protein DIGE is an extremely sensitive method in which a 15 change in protein abundance is more than tw
6. differences The outcome of image analysis is a list of difference protein spots that indicate significant differences between the two protein samples being compared A CRITICAL STEP In general we rely on the two frame looping movies to identify the significant protein changes we want to identify by MS The exogenously added BSA serves as a loading control to balance the image display parameters for making two frame 1356 VOL 1 NO 3 2006 NATURE PROTOCOLS PROTOCOL npg 2006 Nature Publishing Group http www nature com natureprotocols looping movies More precise quantification is done later using SExtractor To accurately assess the level of protein change the Cy3 and Cy5 images are summed and submitted to SExtractor for spot detection which generates a list of elliptical objects that specify individual protein spots This list is used as a mask to determine the protein spot intensities in background subtracted Cy3 and Cy5 images This approach typically yields an SD of 5 7 for unchanging spots Visual difference protein detection requires lt 1 h for an experienced user to complete Computational spot detection methods are becoming more reliable but require manual editing The computational methods are particularly useful when comparing multiple gels Spot picking TIMING 1 2 h depending on the number of protein differences 25 Use the integrated spot cutting tool to click on spots of interest cut a 1 8 mm diameter plug from
7. should be filled with 3 5 l tank buffer and stirred constantly at 4 C in a cold room 21 Electrophorese at a constant current with a maximum voltage set at 500 Typically we set the current at 10 25 mA per gel depending on how long we want the run to take e g 25 mA per gel requires 10 h to complete and 15 mA per gel takes 16h 22 At the completion of electrophoresis remove the gels from the glass plates The stacking gel dye front and IEF strip should be removed and the gel soaked in fixative for gt 2 h with gentle swirling PAUSE POINT Gels can be stored in fixative for several months without significant loss of fluorescent signal For long term storage refrigeration is recommended However protein spots to be identified by MS should be cut out and frozen at 80 C within 1 wk Image acquisition TIMING 30 min per gel 23 Acquire images using your chosen imaging system The DIGE experiment described will yield four images two each from gel 1 and reciprocal gel 2 Each image is 1 024 x 1 280 pixels with a resolution of 135 um per pixel These images should be stored as raw unsigned 16 bit data Image analysis TIMING 1 h per 24 x 18 cm gel with 1 500 protein spots 24 Perform image analysis Image analysis in our laboratory is done using several software applications IPLab Scanalytics QuickTime and SExtractor Visually inspect a two frame looping QuickTime movie of the Cy3 and Cy5 gel images to detect protein
8. to polymerize for gt 8 h if one intends cutting protein spots for MS Unpolymerized acrylamide causes side chain and amino terminal modification of proteins and these might pose problems for MS Gels can be allowed to polymerize overnight They should be layered with sufficient water saturated n butanol that the gels do not dry out It is alright to pour the stacking gel just 4 6 h before the electrophoresis is set up Commercial gels might be suitable provided they do not contain any fluorescent contaminants PAUSE POINT Gels can be stored at 4 C for 2 d provided care is taken to prevent drying Rehydrating the IEF strip TIMING 1 h for set up and 12 h for rehydration 6 Rehydrate Immobiline DryStrips in a DryStrip rehydration tray according to the manufacturer s instructions We prefer to use sample cups to load protein samples We do not recommend including the protein sample in the rehydration buffer as this leads to vertical and horizontal streaks and protein loss A CRITICAL STEP Make sure the IPG buffer matches the pH profile of the IEF strip The Immobiline DryStrips need to be rehydrated in rehydration buffer for 8 22 h Sample preparation TIMING 1h 7 Place the cells or tissue sample in a 1 5 ml centrifuge tube that has a fitted pestle Sufficient material to yield 100 250 ug protein is needed Rinse the cells with an ice cold low salt Na HEPES pH 8 0 buffer that does not contain primary amines Remove excess liquid an
9. AL STEP Wash all IEF accessories on finishing IEF Use double distilled water for washing Wash strip holders with the IEF strip cleaning detergent Use Q Tips Avoid scratching Rinse thoroughly with water and then absolute ethanol Wash electrodes with water and ethanol Nitex strips should be washed with RBS detergent water and finally ethanol Clean strip holder lids and the IPGphor platform PAUSE POINT After equilibration the IEF strips can be stored at 80 C for several weeks Second dimension SDS PAGE TIMING 6 12 h 18 Melt agarose sealing solution in a microwave and place in a beaker of hot water to keep melted Drain the n butanol from the top of the second dimension gel and rinse well with water 19 Remove the IEF strip from the equilibration II solution or thaw if previously equilibrated and frozen Place IEF strip on top of the second dimension gel with the plastic touching the back glass and the acidic end of the strip towards the left Make sure the gel side is not touching the front glass Gently push the IEF strip down until it contacts the stacking gel Make sure not to damage the IEF gel Cover the IEF strip with melted agarose until it just covers the IEF strip Dispense some from each end so that it covers evenly Make sure there are no bubbles If there are bubbles remove them once the agarose solidifies 20 Place the gel in the electrophoresis unit and fill the upper and lower chambers with tank buffer The lower tank
10. HS Amersham Biosciences GE Healthcare Cy5 NHS Amersham Biosciences GE Healthcare Protein clean up kit e g ProteoHook Proteopure Iodoacetamide Sigma e Acrylamide 30 T 2 6 C stock solution Bio Rad N N N N tetramethylethylenediamine TEMED Sigma e Ammonium persulfate APS 10 stock kept at 20 C Sigma IEF strips e g pH 3 10 NL 18 cm Immobiline DryStrips Amersham Biosciences GE Healthcare 3 mm filter paper Whatman Nylon strips 30 130 mesh Nitex screen Sefar America Inc Dimethyl formamide DMF anhydrous 99 9 Aldrich e Methylamine HCl Sigma e Bromophenol blue Sigma e Acetic acid glacial Fisher IPG buffer Amersham Biosciences GE Healthcare e Glycerol spectral or pesticide grade Fluka Standard low M agarose Bio Rad e Methanol HPLC grade Fisher e n butanol Bradford reagent Bio Rad IEF strip cleaning detergent Amersham Biosciences GE Healthcare e RBS 35 detergent concentrate Pierce Ethanol absolute Fisher DryStrip cover fluid GE Healthcare EQUIPMENT IPGphor IEF apparatus Amersham Biosciences GE Healthcare e SE660 vertical gel electrophoresis apparatus Hoeffer Fluorescent gel imager spot picker custom made drawings are available upon request e Model 485 Gradient Former gradient maker Bio Rad DryStrip rehydration tray Amersham Biosciences GE Healthcare Image analysis software applications e g IPLab
11. ariation Hypothetically if one has 10 contamination of neighboring tissue in one sample and 5 variation in another it might lead to artifactual protein differences One way to avoid tissue contamination is to use laser micro dissection which provides a precise method for capturing specific populations of cells Another source of variation can arise during sample clean up or fractionation If sample clean up or fractionation is planned it is best to label the samples independently and then combine them prior to cleanup or fractio nation This will alleviate variation due to handling Simply measuring total protein after sample cleanup or fractionation will not guard against variation in loss of specific proteins during processing The protocol described here outlines the steps we use in performing 2D DIGE experiments We typically use a two dye approach with Cy3 and Cy5 For comparing model systems such as Drosophila or yeast the two dye approach is more reproducible than the three dye method which utilizes a Cy2 labeled pooled sample for normalization The three dye method is better suited to multiple sample comparisons where there is considerable genetic variation as in the analysis of human samples The precursor to the three dye method was a two dye method comparing a Cy3 labeled pooled control test sample and Cy5 labeled control or test samples We always run two gels for each comparison in which the order of labeli
12. d add 100 ul lysis buffer Homogenize the cells with a few passes or turns of the pestle Centrifuge the sample in a microcentrifuge for 5 15 min at 15 000g and 4 C to remove unbroken cells and debris A CRITICAL STEP The majority of our samples are Drosophila embryos which lyse easily by this method Keep the centrifuge tube on ice during homogenization or work in a cold room Some cell types might require more vigorous homogenization such as Saccharomyces cerevisiae which requires vortexing at high speed in the presence of glass beads after addition of lysis buffer 1354 VOL 1 NO 3 2006 NATURE PROTOCOLS PROTOCOL npg 2006 Nature Publishing Group http www nature com natureprotocols A glass homogenizer may be used for larger samples but the lysate needs to be centrifuged at top speed in a microcentrifuge for 5 15 min at 4 C to remove debris 8 Measure protein concentration using a Bradford assay As urea CHAPS and DTT affect the Bradford assay the standards and samples should all be made up in lysis buffer Add 2 ul sample standard to 1 600 ul water in a plastic test tube and mix Add 400 ul Bradford reagent mix and measure the OD at 595 nm within 1 h If the sample is too concentrated dilute with lysis buffer A standard curve should be made with BSA dissolved in lysis buffer at a concentration range of 0 5 to 2 5 mg mlt The blank should be made with 2 ul lysis buffer A CRITICAL STEP Ideally the protein concentration of
13. d online at http www natureprotocols com Reprints and permissions information is available online at http npg nature com reprintsandpermissions 1 O Farrell P H High resolution two dimensional electrophoresis of proteins J Biol Chem 250 4007 4021 1975 2 Scheele G A Two dimensional gel analysis of soluble proteins Characterization of guinea pig exocrine pancreatic proteins J Biol Chem 250 5375 5385 1975 3 Klose J Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues A novel approach to testing for induced point mutations in mammals Humangenetik 26 231 243 1975 4 Gorg A Postel W amp Gunther S The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 9 531 546 1988 5 Hamdan M amp Righetti P G Proteomics Today Protein Assessment and Biomarkers using Mass Spectrometry 2D Electrophoresis and Microarray Technology John Wiley amp Sons Hoboken New Jersey 2005 6 Unlu M Morgan M E amp Minden J S Difference gel electrophoresis a single gel method for detecting changes in protein extracts Electrophoresis 18 2071 2077 1997 7 Lilley K in Current Protocols in Protein Science eds Coligan J E Dunn B M Speicher D W amp Wingfield P T 22 2 1 22 2 14 John Wiley amp Sons Inc New York 2002 8 Gong L et al Drosophila ventral furrow morphogenesis a proteomic analysis Development 131
14. de CCD based fluorescence imager that has an integrated spot picking robot Fig 3 The main issue to consider in gel imaging is that one should try to utilize the full dynamic range of the detector The dynamic range of the detector is measured in terms of the number of discrete grey levels or bits of information that each pixel can accommodate The CCD camera in our imager has a 16 bit dynamic range or 65 536 grey levels Ideally one would like to load enough fluorescently labeled protein and set the exposure parameters so that the bright est protein spot nearly saturates the full dynamic range of the detector Another important issue is the fluorescent background Many of the materials and substances that are placed in the imager can have intrinsic fluorescence For example borosilicate glass is highly fluorescent so any glass used to hold the gels in the imager must be made of quartz or fused silica Although background fluorescent signals can be removed computationally they limit the dynamic range of the gel images and mask signals from low abundance proteins Our gel imaging system is capable of detecting as little as 0 5 fmol of protein and can detect proteins over a gt 10 000 fold concentration range As there are a limited number of such gel imaging systems currently in use we will not describe our imaging protocol here in detail Image analysis in our laboratory is done using several software applications including IPLab Scanal
15. ed to form a pH 3 10 NL strips are a good starting point for whole cell extracts 10 15 gradient gel NL indicates that the strip has a non linear pH gradient with increased sae a ee S AE resolution between pH 5 and 7 The 13 cm strips fit well into the Hoeffer Stock solution Light Heavy Stack SE 660 system that we use for SDS PAGE If using 18 cm strips Acrylamide 30 T 2 6 C 8 25 ml 12 25 ml 400 ul the ends will have to be cut to fit This does not lead to appreciable 1 5 M Tris pH 8 8 6 25 ml 6 25 ml T loss of proteins when using pH 3 10 NL strips as most samples do 0 5 M Tris pH 6 8 _ _ 800 ul not have many proteins focused with pI values near the extremes Cutten T 3 75 A Bae SIG In practice using 18 cm pH 3 10 NL strips improves the resolution o of proteins in the region where most proteins lie pH 4 7 strips can SDS 20 125 ul 125 ul 16 5 ul be used instead of pH 3 10 NL if most proteins are within this pI range H20 10 375 ml 4 175 ml 2 05 ml which is the case for bacterial samples Narrow pH range strips APS 10 82 5 ul 82 5 ul 16 7 ul 1 pH unit are available for closer study of proteins within a region TEMED 8 25 ul 8 25 ul 1 7 ul of interest PROCEDURE Casting the gradient gel TIMING 1 h 1 Assemble the gel cassette using cleaned and dried plates We use homemade 10 15 gradient gels The glass plates used for the SE660 vertical gel electrophoresis apparatus are 24 x 18 cm The spacers used are 1 5 mm in
16. g Step 15 to prevent the IEF strip from drying The IEF gel is fragile and requires careful handling NATURE PROTOCOLS VOL 1 NO 3 2006 1355 npg 2006 Nature Publishing Group http www nature com natureprotocols PROTOCOL 16 Pipette the sample into the sample cup taking care to TABLE 2 IEF program for Step 16 avoid bubbles Place the lid over the strip holder Start the Voltage gradient Duration Duration IEF program shown in Table 2 using 50 uA per strip at 18 C Step Voltage type h kVh We cover the IEF machine with an opaque cover to limit 1 500 Step n hold 1 z photobleaching The time taken to reach 30 40 kVh using 2 4000 Step n hold 1 _ the above protocol is between 6 12 h The duration depends 3 8000 Step n hold 2 4 on the sample and can vary from run to run depending on 4 8000 Step n hold 30 40 the amount of salt and other contaminants in the sample Gels should be equilibrated in equilibration buffer immediately after the run Equilibration of IEF strip for second dimension TIMING 30 min 17 Remove strips and place in 100 mm Petri dishes curled along the inside of the dishes with the gel surfaces facing inward Equilibrate in 10 ml room temperature equilibration buffer I for 15 min with swirling Rinse briefly with water Equilibrate in 10 ml room temperature equilibration buffer II for 15 min with swirling Drain buffer Equilibrated IEF strips can be run on the second dimension immediately A CRITIC
17. its Figure 4 DIGE images a Color overlay of Cy3 green and Cy5 red images of Drosophila embryo extracts Regions of equal Cy3 and Cy5 signals appear yellow b Examples of side by side comparisons of subregions of the DIGE such as ProteoHook or can be broken up by sonication or the addition of nuclease gel shown in a with Cy3 on the left and Cy5 on the right The arrows indicate proteins that have changed NATURE PROTOCOLS VOL 1 NO 3 2006 1357 npg 2006 Nature Publishing Group http www nature com natureprotocols PROTOCOL ANTICIPATED RESULTS There will always be gel to gel variation for 2DE gels This is due to a myriad of uncontrollable features in manufacturing sample preparation and electrophoretic conditions DIGE was developed to circumvent some of this variability The digital imaging system and fluorescence labeling also provide a three or four orders of magnitude linear response which provides much more accurate estimation of protein abundance than silver or Coomassie blue staining of proteins DIGE protein differences can be visualized in a variety of ways including color overlay side by side spot comparison Fig 4a b and two frame looping movies Supplementary Video 1 Supplementary information is available via the HTML version of this article COMPETING INTERESTS STATEMENT The authors declare competing financial interests see the HTML version of this article for details Publishe
18. lysis therefore greatly improves the statistical assessment of proteome variation Here we describe a protocol for conducting DIGE experiments which takes 2 3 d to complete INTRODUCTION The central goals of proteomics include identifying protein changes that differentiate normal and diseased states in cells tissues or organisms and examining how protein changes correlate with developmental age and environment The first stage in comparative proteomics is to separate complex mixtures of protein into indi vidual components this is typically done using gel electrophoresis at the whole protein level or column chromatography at the peptide level Both of these separation schemes have advantages and disadvantages We have focused on two dimensional electro phoresis 2DE because of its accessibility to most laboratories This approach was described simultaneously by several groups in 1975 refs 1 3 Despite the substantial advances in the technology since its launch the most notable of which was the introduction of immobilized pH gradients in the first dimension some of the more significant systemic shortcomings have remained unsolved The most troublesome of these is the inherent lack of reproduci bility between gels Efforts to surmount this limitation have mostly focused on developing computational methods for gel matching These approaches have had limited success because the sources of gel to gel variation are numerous complex
19. ng is reversed referred to as reciprocal labeling This allows one to differentiate between sample dependent differences and rare dye dependent differences The latter are presumably due to incomplete solubiliza tion of proteins allowing the two dyes to associate differently with the protein Loading equal amounts of each protein sample is advisable but high precision is not required as slight load differ ences are normalized during image analysis All steps should be performed on ice or in a cold room if necessary As soon as lysis is complete samples can be stored at 80 C in aliquots of 100 250 ug at concentrations gt 1 mg ml When comparing whole cell extracts we generally load 80 200 ug of each sample 160 400 ug total protein High quality digital imaging of DIGE gels is essential for detecting proteome changes To peer as deeply into the proteome as possible it is important to use an imager that is capable of true 16 bit data collection Fluorescent gel imagers come in two formats scanner based or CCD camera based There are several commercial gel imagers such as the Typhoon imager Amersham Biosciences GE Healthcare which is commonly used for DIGE gel imaging The Typhoon imager is based on a scanning laser illumination system and photomultiplier detector The 1352 VOL 1 NO 3 2006 NATURE PROTOCOLS gel scanning systems that are suitable for DIGE analysis require a different device for spot picking We use a homema
20. npg 2006 Nature Publishing Group http www nature com natureprotocols PROTOCOL Two dimensional difference gel electrophoresis Surya Viswanathan Mustafa nl amp Jonathan S Minden IDepartment of Biological Science Carnegie Mellon University Mellon Institute 4400 Fifth Avenue Pittsburgh Pennsylvania 15213 USA 2Present address Proteopure Incorporated 900 William Pitt Way Pittsburgh Pennsylvania 15238 USA Present address Bogazici University Department of Molecular Biology and Genetics Bebek 34342 Istanbul Turkey Correspondence should be addressed to J M minden cmu edu Published online 26 October 2006 doi 10 1038 nprot 2006 234 Two dimensional difference gel electrophoresis 2D DIGE is a modified form of 2D electrophoresis 2DE that allows one to compare two or three protein samples simultaneously on the same gel The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis Proteins that are common to the samples appear as spots with a fixed ratio of fluorescent signals whereas proteins that differ between the samples have different fluorescence ratios With the appropriate imaging system DIGE is capable of reliably detecting as little as 0 5 fmol of protein and protein differences down to 15 over a gt 10 000 fold protein concentration range DIGE combined with digital image ana
21. o standard deviations SDs above the normal variation One must take great care in deciding which samples to compare while bearing in mind the sources of variation If specific tissues are to be compared one must carefully dissect Control cells Test cells Prepare protein extracts gt TON fo E Label with ma Cy5 NHS Mix samples and run on 2D PAGE Image with MWt fluorescence gel imager pl Figure 1 Schematic of DIGE analysis Extracts are made of two cell samples denoted A and B These extracts are separately labeled with Cy3 NHS and Cy5 NHS which covalently link to lysine residues A low stoichiometry of labeling is used where 5 of all proteins carry a single dye molecule The labeled protein extracts are then combined and co electrophoresed on a 2DE gel The gel is then imaged on a fluorescent gel imager at the Cy3 and Cy5 wavelengths MWt molecular weight pI isoelectric point NATURE PROTOCOLS VOL 1 NO 3 2006 1351 npg 2006 Nature Publishing Group http www nature com natureprotocols PROTOCOL ILA aO CH 4 CHa COR CH tie Cy3 Cy5 R NHS Figure 2 Chemical structure of DIGE dyes Propyl Cy3 NHS and methyl Cy5 NHS are shown here These compounds are charge and mass matched The fluorescent characteristics of Cy3 and Cy5 are dictated by the three carbon and five carbon polyene chains respectively linking the two indoline rings the tissue to avoid v
22. pass filters Chroma Technology PROTOCOL npg 2006 Nature Publishing Group http www nature com natureprotocols as that achieved with DeCyder None of the current image analysis applications are completely automatic in that the computer deter mines which protein spots are changing and the degree of change A certain amount of editing of the detected spots by visual inspection is required We find that visual inspection of a two frame looping movie of the Cy3 and Cy5 gel images is the most reliable method for detecting protein differences and for spot list editing For visual inspection we find that two frame looping movies are much more robust than superimposing two pseudocolor images of Cy3 labeled and Cy5 labeled proteins While state of the art automated spot detection applications are becoming more robust visual inspection is still used as the final arbiter The outcome of image analysis is a list of difference protein spots that indicate putative differentially expressed or modified candidate proteins This spot list can be used for large scale protein profiling studies using standard bioinformatics tools Despite DIGE being sensitive and reproducible three caveats need to be mentioned First 2DE does not efficiently resolve integral membrane proteins This is due to their hydrophobic domains causing precipitation during isoelectric focusing IEF Other laboratories are working to solve this problem Second labeling wi
23. phenol blue and 1 wt vol IPG buffer Use the appropriate IPG buffer that corresponds to the pH range of the IEF strips in the experiment Store rehydration buffer at 4 C Use within 7 d Equilibration stock 50 mM Tris pH 8 8 6 M urea 30 vol vol glycerol 2 wt vol SDS and 0 002 wt vol bromophenol blue Can be stored at 4 C for 4 6 wk Equilibration buffers I and II Prepare equilibration buffers I and II as needed using 10 ml per IFF strip equilibration buffer I comprises 1 wt vol DTT in equilibration stock equilibration buffer II comprises 2 5 wt vol iodoacetamide in equilibration stock Gradient gel solutions See Table 1 for the composition of the three acrylamide solutions needed to form a 10 15 gradient gel that is 14 x 24 cm These solutions can be stored without SDS APS and TEMED for 2 3 wk at 4 C Tank buffer 25 mM Tris 190 mM glycine and 0 1 wt vol SDS Can be made or purchased as a 10x stock Agarose sealing solution 125 mM Tris pH 6 8 0 1 wt vol SDS 1 wt vol agarose and 0 002 wt vol bromophenol blue Heat in a microwave until the agarose dissolves Fixative 40 vol vol methanol HPLC grade and 1 vol vol acetic acid glacial NATURE PROTOCOLS VOL 1 NO 3 2006 1353 npg 2006 Nature Publishing Group http www nature com natureprotocols PROTOCOL IEF strips IEF strips are available in different pH ranges and sizes TABLE 1 Composition of acrylamide solutions need
24. ression experimental design using minimal CyDyes Proteomics 5 3105 3115 2005 Tonge R et al Validation and development of fluorescence two dimensional differential gel electrophoresis proteomics technology Proteomics 1 377 396 2001 Yan J X et al Fluorescence two dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli Proteomics 2 1682 1698 2002 Bertin E amp Arnouts S SExtractor software for source extraction Astron Astrophys 117 393 404 1996 Suehara Y et al Proteomic signatures corresponding to histological classification and grading of soft tissue sarcomas Proteomics 6 4402 4409 2006 Shaw J et al Evaluation of saturation labelling two dimensional difference gel electrophoresis fluorescent dyes Proteomics 3 1181 1195 2003 Zhou S Bailey M J Dunn M J Preedy V R amp Emery P W A quantitative investigation into the losses of proteins at different stages of a two dimensional gel electrophoresis procedure Proteomics 5 2739 2747 2005
25. th the amine reactive DIGE dyes limits one to sub stoichiometric labeling also known as minimal labeling where less than 5 of all proteins carry a single bound dye molecule and the rest have no bound dye For proteins that are gt 25 kDa there is no appreciable molecular weight shift between labeled and unlabeled protein while there is a slight but predictable shift for smaller proteins in which unlabeled proteins run about half a spot diameter faster than their labeled counterparts This shift problem has been addressed with the development of cysteine reactive dyes These dyes allow one to saturation label all available cysteines which eliminates the shift between labeled and unlabeled proteins as all proteins are maximally labeled Third there is a lower limit to the amount of protein required for mass spectrometry MS which is 10 fmol for the average mass spectrometer Again other laboratories are striving to lower the MS detection limit Regardless of these limitations DIGE com bined with MS is a sensitive robust and useful approach for comparative proteomics MATERIALS REAGENTS A CRITICAL Use double distilled H O unless otherwise stated A CRITICAL The source and purity of ingredients is critical especially for solutions used for labeling and IEF e Urea Bio Rad Thiourea Sigma e CHAPS Roche DTT Sigma e SDS 20 stock solution Bio Rad e HEPES 100 mM Na HEPES pH 8 0 stock Sigma e Cy3 N
26. the gel and deposit it in a 96 well plate designed for a protein digestion robot ProGest The wells of the receiving plate should be filled with 1 vol vol acetic acid The imager software allows one to create a list of protein spots to be retrieved After all the desired spots are cut the acetic acid solution should be removed and the plate moved to the digestion robot A CRITICAL STEP A key difference between the gel imaging system we use and the Typhoon imaging system is that we use an open gel format that is amenable to imaging and spot picking within the same device whereas the Typhoon imager is best suited to imaging the gel within the electrophoresis plates so spot picking is done on a different device The open gel format does not require the gel to adhere to one of the two electrophoresis plates E PAUSE POINT The cut gel fragments can be stored after acetic acid removal at 80 C for at least several weeks Protein identification 26 Identify the difference proteins using MS This can be done by routine methods using a variety of instruments DIGE labeling with Cy3 NHS or Cy5 NHS labels only a small fraction of proteins in the lysate 5 of all proteins have a single dye molecule bound whereas the rest have none Hence DIGE labeling does not affect the MS identification of proteins It is important to note however that the DIGE imaging system is capable of detecting lt 0 5 fmol of protein This is well below the limit of identifica
27. the lysate should be 1 2 mg mlt If the lysate concentration is too high dilute with lysis buffer If the concentration is too low the sample may be concentrated by precipitation but this could create problems with protein loss during precipitation re solubilization and pH adjustment It is preferable to start with sufficient material to yield a suitably concentrated lysate Another important consideration is the removal of contaminating factors that affect IEF such as salt lipids detergents and nucleic acids There are a number of commercial protein clean up kits that are worth testing however one must be cautious about protein loss and pH effects We have tested several protein clean up kits and found the ProteoHook reagent to be the best at maintaining the relative proteome composition and to be the most reproducible We recommend labeling the protein samples first and then combining the samples before using a protein clean up protocol Protein labeling TIMING 1h 9 Steps 9 12 describe a typical DIGE experiment in which protein samples denoted A and B are to be compared Set up four 1 5 ml microcentrifuge tubes numbered 1 4 Pipette 80 ul protein sample A which contains 80 200 ug of protein in lysis buffer into tubes 1 and 3 Pipette 80 ul protein sample B which contains 80 200 ug protein in lysis buffer into tubes 2 and 4 Try to use equal amounts of protein in each sample Optionally you can also add 1 ul of 1 mg ml
28. thickness One can bypass this step by purchasing pre made gradient gels for 2DE 2 Set up a standard two chamber gradient maker so that the outlet tube directs the acrylamide solution into the top of the gel cassette CAUTION Acrylamide is a neurotoxin wear gloves and use appropriate handling precautions A CRITICAL STEP Make sure the opening of the outlet tube is sufficiently wide that the pouring process takes lt 2 3 min otherwise the gel might begin to polymerize during the pouring process We use a disposable 200 ul pipette tip cut at an angle which fits snugly between the glass plates and allows for rapid casting of the gradient gel 3 Add SDS to the heavy and light acrylamide solutions and mix well Then add 10 wt vol APS and TEMED when ready to pour the gel 4 Pour heavy and light solutions simultaneously into the front and back chambers of the gradient maker respectively Open the mixing channel and the front pinchcock simultaneously to start pouring the gel Overlay with 750 ul water saturated n butanol top layer and allow the gel to polymerize for gt 1 h If pouring several gels at a time rinse the gradient maker with water between gels A CRITICAL STEP Use a magnetic stirrer and stir bar in the front chamber to ensure sufficient mixing of the heavy and light solutions 5 Drain off the n butanol and rinse with water to remove Pour the stacking gel and overlay with n butanol A CRITICAL STEP Gels should be allowed
29. tion of peptides derived from gel spots using MS which is 10 50 fmol To identify low abundance difference proteins one must either enrich for the proteins of interest or pool protein from multiple DIGE gels TIMING Casting the gradient gel 1 h Rehydrating the IEF strip 1 h for set up and 12 h for rehydration Sample preparation 1 h Protein labeling 1h First dimension IEF 6 12 h Equilibration of IEF strip for second dimension 30 min Second dimension SDS PAGE 6 12h Image acquisition 30 min per gel Image analysis 1 h per 24 x 18 cm gel with 1 500 protein spots Spot picking 1 2 h depending on the number of protein differences TROUBLESHOOTING There are two main reasons for poor results insufficient protein labeling and incomplete IEF Insufficient labeling is commonly due to the pH of the lysate being lt 8 0 or the presence of excess primary amines such as Tris If there is sufficient sample one can test the pH using a small piece of pH paper Tris lt 50 mM does not appear to interfere with protein labeling and 30 mM is recommended Incomplete focusing is usually the result of interfering contaminants such as salts and nucleic acids Salts which can be removed using a protein clean up kit prevent the IEF from reaching the desired voltage Nucleic acids make the sample viscous and difficult to handle and also interfere with focusing Nucleic acids can be removed by commercially available clean up k
30. ytics QuickTime and SEx tractor There are integrated image analysis packages for DIGE gels the most commonly used of which is DeCyder Amersham Biosciences GE Healthcare We use IPLab and QuickTime for image visualization and annotation SExtractor is an astrophysics freeware application that has been adapted to quantify protein spot intensities and ratios https sourceforge net projects sextractor Although these separate applications are more time consuming than the commercial packages we find the analysis to be as reliable CCD camera Gel cutter stage Stepping motor Cutting tip 96 well plate Light tight cabinet Y axis stage X axis stage Figure 3 Fluorescence gel imager spot picker This diagram illustrates the components of the DIGE gel imager This device comprises the following components a scientific grade Peltier cooled 16 bit CCD camera Photometrics Roper Scientific an 105 mm macro lens Nikon a computer driven stage New England Affiliated Technology and a spot picker drive Applied Precision All electronic components are controlled by a computer workstation running Windows and custom software Not shown is the illumination system which is composed of two 250 W quartz tungsten halogen light sources Oriel that direct their light through motorized filter wheels Ludl The light is directed onto the gel via fiber optic light guides Oriel and fluorescence wavelengths are selected by band
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