Short instructions for CIAN DiGE users
Contents
1. Please see the DIGE manual for detailed instructions Here are a few key points Standard labeling reaction 50ug protein 400pmol CyDye according to several unprinted sources and our own experience half the amount of dye is also sufficient to give a good signal Standard labeling conditions should be re evaluated for samples of lower complexity reduce amount of total sample or samples containing one or several overabundant proteins increase amount of sample to see low abundance proteins or if you know that you are dealing with something that is unusual in its lysine content The protocol calls for making a 1nmol ul concentrated stock and then a 400pmol ul working solution of the dyes the latter can t be stored for more than a week It is also possible to set up the labeling reactions with the concentrated stock directly A slightly basic pH is crucial for labeling Check the pH of your sample by spotting one ul on pH paper at least one time for a typical sample to make sure your buffer maintains the pH in the presence of your proteins DMF for dye dilution should be purchased fresh for each batch of dye to minimize exposure to humidity A 10mM lysine solution for quenching is in the fridge Prepare fresh from powder if desired Labeling workflow put measured protein amount in clean Eppendorf tube add appropriate amount of dye solution mix spin down incubate 30min on ice in the dark quench by adding 1ul of 12. on short end of gel sandwich metal parts will be on the bottom carefully lower gel sandwich onto scanner platen avoid water or buffer getting under glass sandwich note orientation use DiGE template in control software important settings are acquisition mode fluorescence tray DiGE Ettan DALT select 1 or 2 gels focal plane 3mm press sample on DiGE naming format Dye Emission filter Laser Cy2 520 BP 40 Blue 488 Cy3 580 BP 30 Green 532 Cy5 670 BP 30 Red 633 prescan at 1000um resolution to adjust PMT voltage start out around 500V final scan at 100um resolution Note on adjustment of PMT voltage Intensity values can maximally be 100K The values from pre scans tend to be 7K lower than in the high resolution scan Aim for a maximum of 75K check in ImageQuant in the pre scan to avoid oversaturation in the final scan For more details see CIAN Typhoon mini manual Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 7 Post staining 10 Before starting a staining protocol clean containers locate reagents Read detailed protocols for specific instructions this is only an overview Deep Purple total protein stain All steps at RT with gentle shaking protect stain stained gel from light required volume per gel min 300 500ml 11 for fix wash Fixation A 1 citric acid 1 5h or over night 15 ethanol Stain B 1 200 thawed conc
3. 29 2 ml 37 5 ml 1 5M Tris pH8 8 62 8 ml 87 9 ml 113 ml Milli Q H20 85 3 ml 119 4 ml 153 5 ml 10 SDS 2 5 ml 3 5 ml 4 5 ml 10 APS fresh 1 0 ml 1 4 ml 1 8 ml TEMED 125 ul 175 ul 225 ul Total 250 ml 350 ml 450 ml Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 8 Preparation of DALTsix apparatus get out and clean all parts of the DALTsix prepare 5I of 1x SDS PAGE running buffer for lower tank anode and 1 21 of 2x SDS PAGE running buffer or 1x buffer containing 2x the amount of SDS by adding 1 100 volume of 10 SDS stock for upper tank cathode assemble tank fill with 41 lower tank buffer switch on buffer circulation pump by plugging in black cable switch on Multitemplll water bath adjust temperature see below prepare or melt agarose sealing solution 1 LMP agarose in upper tank buffer 1 2ml per gel let cool to 60 C in heat block get out and clean outsides of gels rinse sample well with running buffer Equilibration of IPG strips thaw equilibration buffer stock at room temperature for each 24cm strip prepare 10ml of equilibration buffer 1 5mg ml DTT 50mg 10ml and 10ml of equilibration buffer 2 45mg ml iodoacetamide 450mg 10ml disolve by shaking at RT equilibrate strip for 15min each in buffer 1 and 2 at RT with gentle shaking Loading of strips on gels place gel on bench long plate on bottom wet strip with running buffer place strip on long edge of pla
4. maximum follow up fixed and or stained gels can be kept several days weeks in the fridge Spot picking time varies depending on technique used hand picking robot follow up picked spots can be kept for several days weeks in appropriate buffer before submitting to mass spectrometry Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 3 2 General considerations Sign up online for use of the first and second dimension separation equipment If you don t need a booked time slot cancel your reservation Let Elke know of your plans to run a large 2D or DiGE gel well in advance so that all supplies can be ordered and received Typical order to delivery times in business days without backorder issues DiGE CyDyes 2 10 days IEF strips 1 3 days if in stock but can be 1 2 weeks pre cast gels at least 1 week depending on supplier Note on the IEF strip inventory sheet on the freezer how many strips you have used and at what date That way our inventory should always be current The lab is based on a clean before and after policy please clean the equipment you are using to your specifications before use and clean up after yourself and put equipment into storage condition after you are done Weare always trying to improve so please let us know of any suggestions Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 4 3 DiGE CyDye labeling
5. post stained gel image has to be loaded matched and annotated in Decyder before submitting for picking Stain gel silver or SyproRuby DeepPurple for floating gels SyproRuby or DeepPurple for backed gels Scan or image gel For loading into Decyder first make copy of your BVA workspace into a new project using the Organizer from the Decyder main menu screen to make sure you carry over all matching information and annotations but not to destroy the analysis should things not work as planned o Load gels into new project containing copied BVA workspace o Perform spot detection in DIA o Open copy of BVA workspace add pick gel image edit add template DIA workspace to match put master Cy2 image and pick gel image into windows create a few landmarks use match primary option when matching that way all the other matched images stay as they are check matching especially of the pick list spots Create images to use for picking only of the post stained gel with and without spot outlines and annotations See document CIAN Viewing and Editing of Typhoon Images section VII for details Gel or gel pieces submitted to MS facility should be in ultrapure water or 1 acetic acid Check with your MS facility contact about the specific requirements Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 13 10 Recipes of solutions provided in the CIAN DiGE lab based on rec
6. raw data DIA spot detection and first in gel analysis for the images from each single gel e create workspace a DIA workspace is a folder that contains the DIA analysis data for one gel it can be filed in the same project folder as the image data or a new project can be generated e select gel images to be analyzed remember only the images of a single gel can be analyzed at one time e process process gel image you may change estimated number of spots to match expectations then look at the results in the scatter plot adjust contrast and brightness of the image but no further steps are necessary to proceed to BVA e save workspace for use in BVA BVA analysis of DIA spot maps of all gels in one DIGE experiment merges gel spot maps normalizes intensities statistical analysis t test ANOVA e create workspace a BVA workspace is a folder that contains the BVA analysis data for a DIGE experiment it can be filed in the same project folder as the image and DIA data or a new project can be generated e from the menu select DIA workspaces to be included in this analysis e assign images to groups in Experimental Design View in the upper corner of the ST spot map table screen the images of the internal standard should already be assigned to a group named Standard e define landmarks in MT match table display standard images of all gels check landmark mode click on landmark spots in master image and then all t
7. 0mM lysine mix incubate 10min on ice in the dark Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 5 4 First dimension IEF Rehydration loading get out thaw sample sample buffer DTT MW 154 2 or DeStreak solution clean coffin s with Strip Holder Cleaning Solution and toothbrush let drain and air dry or blot dry with Kimwipes clean two pairs of forceps prepare some Kimwipes on work surface to put down strip if needed combine sample sample buffer IPG buffer and DTT to make the appropriate rehydration volume for the strip used if using DeStreak reagent omit DTT add DeStreak to 12ul ml i e for 24cm strip use 5 4ul IPG strip length Total volume per final conc final conc strip 10mg ml DTT 1 IPG buffer 13 cm 250 ul 2 5 mg 2 5 ul 16 2ul of 1M 24 cm 450 ul 4 5 mg 4 5 ul 29 2ul of 1M use of a 1M stock made in water will dilute the other components of the rehydration solution so adding the solid is preferred _if more than one sample note sample ID coffin ID and strip ID on a piece of paper DO NOT write on strips or coffins as lab markers contain fluorescent components the printed number on the strip will be visible on your DiGE scan pipet sample into middle of coffin try to avoid bubbles get strip from 20 C freezer sign out at freezer door remove from package peel off protective foil using forceps starting at a
8. 2D Gel Electrophoresis for DIGE at CIAN NOTE This document is a collection of short protocols for running 2D DiGE experiments at the CIAN DiGE lab It is meant as an introduction and overview for the beginner and a quick reference for more experienced users It is NOT sufficient to read this alone you will need to refer to more comprehensive resources and instrument manuals for details of the procedure last updated April 2013 Elke Kuster Schock Content A aA ING oct tetas colts ES AEA aden E E AAEE 2 2 general considerations iacecre aloe tt css lated is ee ee ie ier eet cian ae rote 3 Oty GY DVS JAD SIMO ts errre arire reeves ere enet ee ee Ee e Oe E EE eae uccengsem rene 4 4 first dimension separation isoelectric focusing IEF eeeeeeeeeeeeeeeeeeteteeeees 5 5 second dimension separation SDS PAGE cccccceeeeeecceeeeeseeeeeeeeeenaeeeeeeeeees 7 6 DIGE g l SCA acute a ead cies Ares a ata ES Cor oa ees ea teat 9 Ta POSI SIAINING eee ee ieee Re ee 10 8 data analysiS with DeCyder mst jceccscu cece iii aenean eeeve bh cedetit ages 11 9 spot PICKING WOPKTIOWE cvcesscevesrcacsacees gecieestaaieeis cards peateadaatadecaidachsasstaasadaadeigabiascexs 12 10 recipes of solutions provided in the CIAN DiGE lab ccecceeeeeeeeeeeeeeeeeees 13 i B 0 EIE a ASEA AE E 14 CIAN 2D Gel Electrophoresis for DiGE 2 1 Time line excluding data analysis Sample preparation time variable dep
9. cidic anodic end basic cathodic end is softer and stickier lower strip onto sample in coffin gel side down writing is right side up with end towards pointed end of coffin remove bubbles and distribute sample by carefully lifting and lowering strip from its ends and sliding it back and forth overlay strip with 1 2ml of DryStrip Cover Fluid mineral oil using disposable Pasteur pipette or blue tip close lid bubbles in oil are not detrimental place coffin on IPGphor in the designated orientation and position space coffins evenly if using only one coffin place another empty coffin on IPGphor place lid adaptor onto coffins close PGphor lid Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 6 start IPGphor power button back left select program for active rehydration don t use the rehydration section but program it as a first step using 30 50V at 20 C for at least 10h Isoelectric Focusing after Rehydration Loading end and exit rehydration program hit stop button 3 times on a clean surface dampen electrode wicks with 7ul water or 50mM DTT 7 7mg ml respectively or dip in and blot dry using forceps slip H20 wick under end DTT wick under end of strip so that it is between gel and electrode refill oil if needed replace coffin lid put coffin s back on IPGphor if you want the computer to log your run start IPGphor cont
10. ending on experimental system follow up samples can be stored frozen in sample buffer CyDye labeling labeling time 1h follow up load immediately on IEF strip Sample loading and strip rehydration preparation time 1h rehydration time at least 10h or over night follow up IEF has to be carried out immediately after rehydration First dimension separation IEF preparation time lt 30min if doing active rehydration separation time variable depending on strip type length sample always several hours should be monitored during last step follow up immediately load strip on second dimension gel or freeze at 80 C for up to several days weeks Second dimension separation SDS PAGE preparation time if self cast gels add casting time 1h polymerization over night ie casting should be done in parallel to rehydration IEF strip equilibration loading starting run 1 2h separation time variable 5 7 hours can be run over night follow up for DiGE scan immediately for staining or storage fix immediately DiGE scan preparation time disassembling gel tank and cleaning plates 1h scan time prescan to adjust parameters 30min actual scan per gel with three fluorophores 40min follow up fixing for post staining or storage if no picking planned just clean up Post staining preparation time 1h fixation and staining min several hours fixation or staining can be done over night except for backed gels 1 5h
11. entrate diluted in 1 5h 100mM sodium borate pH10 5 Wash C 15 ethanol 30min Acidification A 1 citric acid 30min up to over night 15 ethanol to reduce background Rinse C 15 ethanol 5min before imaging Storage D 1 citric acid Scan on Typhoon excitation green laser 532nm emission pre scan 1000um resolution scan 100um resolution S60LP or 610BP Silver stain compatible with mass spectrometry see separate protocol ProQ Diamond Phosphoprotein stain see manufacturer s instructions use before total protein stain if desired Sypro Ruby total protein stain All steps at RT with gentle shaking protect stain stained gel from light required volume per gel 300 500ml Fixation 7 acetic acid 30min repeat 50 methanol Stain Ready to use stain over night Wash 7 acetic acid 30min 10 methanol Rinse Water 5min repeat Scan on Typhoon excitation blue laser 488nm emission 610BP pre scan 1000um resolution scan 100um resolution Elke Ktister Sch ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 11 8 Data analysis with DeCyder See below for introduction use manual for details DeCyder 6 5 overview and very short instructions Gel Loader loading the image data from the Typhoon scanner into the Oracle database for use in DeCyder only done once images can be used for multiple analyses by creating new projects workspaces that point to the same
12. he other images e Match gels e Check matches in MT image view display all Standard images and look at match vectors in zoom if needed break or add matches rematch e In PT protein table view perform protein statistics calculations e Check potential candidate spots individually EDA extended data analysis of one or several BVA workspaces unsupervised and supervised clustering algorithms heat maps SOM k means Principle Component Analysis short instructions not available Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 12 9 Spot picking workflow If picking from DiGE gels treat samples and gels with care to avoid keratin contamination keep handling to a minimum wear protective clothing at all times Consider loading extra unlabeled protein along with CyDye labeled samples to increase the amount of protein in each spot Check with MS facility about their staining preferences Adjust protocol accordingly After scanning gels open glass plates carefully with wedge tool fix gels in fixative appropriate to downstream stain Store gels either in fixative or in 1 acetic acid or in ultrapure water at 4 C cold room while you do the Decyder analysis When happy with pick list contact MS facility to make appointment for sample or gel drop off specify number of spots to process Time a fluorescent post stain to be fresh for robotic picking keep in mind that the
13. ipes in the GE DiGE manual SDS Equilibration Buffer Stock Reagent Quantity Final concentration Tris 1 0M pH8 0 20ml 40ml 100mM Urea MW 60 06 72 07g 144 14g 6M Glycerol 99 5 v v MW92 09 60ml 75 6g 120m1 151 2g 30 v v SDS MW288 33 4g 8g 2 w v Bromophenol Blue 1 stock 400ul 800ul 0 002 w v H20 MilliQ to 200ml to 400ml Rehydration Buffer Reagent Quantity Final concentration Urea MW 60 06 10 5g 7M Thiourea MW 76 12 3 8g 2M CHAPS MW 614 89 1g 4 w v Bromophenol Blue 1 stock 50ul 0 002 w v H20 MilliQ to 25ml SDS PAGE Running Buffer 10x Reagent Quantity Final concentration Glycine MW 75 07 1152g 1 92M Tris MW 121 1 242g 250mM SDS MW 288 38 80g 1 w v H20 destilled RO to 8l Elke Kister Sch ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 14 11 Sources Bibliography personal communication Jackie Vogel Hong Han Renee Wang Fr d ric Dallaire Madeleine Pool Yovany Moreno Wissal El Assaad all McGill University David Friedman Vanderbilt University Nashville TN USA Tracy Andacht University of Georgia Athens GA USA company manuals handbooks Ettan DiGE System User Manual GE Healthcare 2005 2 D Electrophoresis Principles and Methods GE Healthcare 2004 manuals for IPGphorll Immobiline DryStrip IPG strips DALTsix MultiTemplll Powersupply EPS 601 Deep Purple stain Ty
14. phoon trio scanner ImageQuant software Decyder software all GE Healthcare Sypro Ruby stain ProQ Diamond stain Rhinohide Invitrogen Molecular Probes pdf versions of most manuals available from the company websites or from Elke textbook R Westermeier T Naven H R Hopker 2008 Proteomics in Practice A Guide to Successful Experimental Design Wiley VCH papers S Viswanathan M nl JS Minden 2006 Two dimensional difference gel electrophoresis Nature Protocols 1 3 1351 8 protocol style overview of the technique from the inventors NS Tannu SE Hemby 2006 Two dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling Nature Protocols 1 1732 42 another protocol more focused on GE products other sources GE Healthcare 2D Electrophoresis Discussion Board http life sciences forums com Anyone can read and post on 2D related issues replies provided by forum members some of whom are experts in the field Elke Kuster Sch6ck last updated April 2013
15. rol software on Typhoon computer leave on during run save logfile after run ends check edit program start IEF program see hints below clean equilibration tubes allow to dry before use at the end of the program take strips out of coffins blot oil from back and sides of strip on Kimwipes and place strips one equilibration tube each freeze rapidly at 80 C for storage or proceed directly to second dimension IEF program hints too many variables to make rules use the manufacturer s leaflet that came with your strips as a guideline check the recommendations in the GE 2DE Handbook p 65 ff the high voltage in the final step s e g 8000V might only be reached incrementally or not at all depending on the sample conductivity for consistency among runs the length of this step should be limited by Vh not time if you might not be around when the program ends add a step at the end of your program at 500V for several hours to avoid the proteins diffusing from their positions try to keep this to a minimum and add the total accumulated kVh at the end of the run to your notes Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 7 5 Second dimension SDS PAGE on Ettan DALTsix Casting single percentage gels with gel strengthener clean gel caster black wedge funnel separator sheets thin and thick dummy plates glass plates use RO water and lint free wipes assemble gel caster lying fla
16. t start with thin separator sheet then glass plates or dummy thin separator etc until six gel cassettes or dummies are assembled finish with the last thin separator and fill the gap with as many thick separators as needed make sure the foam seal on the face plate is lubricated lightly GelSeal and sits well in the groove turn two screws into threaded holes for two or three full turns place face plate on caster so that notches are resting on screws clamp sides with six spring clamps then hand tighten screws place assembly upright onto bench pad check that it is level prepare 0 1 SDS solution in plant sprayer at least 50ml per gel prepare acrylamide mixture fresh APS solution or add as solid after adding catalysts mix by swirling start casting rapidly using funnel in filling channel in the back until it is about 1cm below the top of the short plate overlay with 0 1 SDS by spraying exhaustively along top from about 30cm away or water saturated butanol via pipette change to 0 1 SDS or Milli Q water after 3h to avoid corrosion of plastic parts on the caster wrap caster in plastic wrap and allow to polymerize at RT over night remove gels and clean off any polymerized acrylamide clean caster use right away or wrap in foil and store in container at 4 C keep moist with running buffer or 0 1 SDS Recipe for 12 5 homogeneous gels 2 gels 4 gels 6 gels 40 acrylamide bis 37 5 1 77 8 ml 108 9 ml 140 ml Rhinohide 20 8 ml
17. te gel side up acidic end to the left of long plate place gel upright into rack push backing of strip down onto gel surface with a thin spacer or ruler avoid trapping bubbles seal strip into place by slowly pipetting agarose onto both ends of the strip Running gels place gels into slots in anode assembly fill empty slots with dummies slide upper buffer chamber over gels lubricate with upper buffer if needed fill upper buffer chamber to between marks top up lower buffer to same level using funnel close lid cover gel tank with black plastic bag for DiGE start run standard conditions 25 C step1 1W gel 60min step2 13W gel until blue front leaves gel about 5 6 hours overnight 30 C step1 1W gel 60min step2 2W gel 16h Elke Kister Sch6ck last updated April 2013 CIAN 2D Gel Electrophoresis for DiGE 9 6 DiGE gel scan Gels have to be scanned right after the second dimension separation If running three or more gels scan two at a time and leave the rest in the gel tank under a very low voltage or wrap in plastic foil and store at 4 C in the dark Allow to warm to room temperature before scanning make sure that scanner is on start Typhoon control software clean scanner platen with 70 ethanol 10 H2Oz last with water take gels out of the tank rinse glass plates clean with distilled water dry with Kimwipes place long gel alignment guide onto Typhoon scanner put gel gripper
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