HPE™-FlatTop Tower - SERVA Electrophoresis GmbH
Contents
1. 9 Grip the gel surface up at the two lateral edges at the protruding film bend into a U shape and slide the film backing left and right on the cooling plate to distribute the cool con tact fluid evenly Fig 4 4 10 Place the gel onto the cooling plate the IPG strip slot towards the cathode the cathodal edge of the IPG strip slot matching line 15 11 Remove excess cooling fluid along the film edges with lint free paper tissue 12 Place the electrode wicks onto the gel edges overlapping them by at least 2 mm Hold wicks horizontally never at an angle as this causes unequal buffer concentration along the wick Smooth out air bubbles with a roller dl aE 14 Apply 5 ulSDS marker proteins to the marker well s 13 Trim the film support of the IPG strips on both sides Place the IPG strips gel side down anodal sides to the right into the slots of the SDSGel and push them carefully towards the anode edges of the slots fig 4 5 Gently slide along the backing of the strips with the forceps to ensure good contact to the bottom of the slots 15 Close the lid while lowering the electrodes on the wicks plug in the cables switch the valve to cooling 15 C switch on the FlatTop Tower main switch pump and electronic control and start the run according to table 4 2 16 After 1 hour 10 min interrupt the run press wait on the powers supply remove the IPG strip s and then continue the run2. Running conditions See table 4 2 for the maximum settings Table 4 2 Running conditions 15 C Steps Voltage Current Power Time S1 100 V 7mA 14mA 21mA 28 mA 1W 2W 3W 4W 30 min i tt Pi Ti vi n i tT S2 200V 13mA 26mA 39mA 52mA 3 W 6 W 9 W 12 W 30 min f i TT tt tt tt th TT S3 300V 20mA 40mA 60mA 80mA 5W 10 W 15W 20W 10 min je t i f f i n n T after this step remove the IPG strips S4 1000V 40mA 80mA 120mA 160mA 25W 50W 75W 100W 3 h 00 min is f i f f tt TT vr m valid for homogeneous gels for the gradient gel 10 15 this step 4 takes 3 5 h For programming BioRad Power Supplies only Set as constant tr Set as limit 14 5 Two Dimensional Electrophoresis Large Gels Always wear powder free disposable gloves Important Only use the SERVA buffer kit for the running buffers and equilibration solutions 1 Apply 45 ml of each electrode buffer to the respective electrode wick in the compartments of the SERVA PaperPool fig 5 1 distribute the solution evenly with a roller and soak for a minimum of 15 minutes Fig 5 1 Soaking electrode wicks in 45 ml electrode buffer 2 Prepare the two equilibration solutions from the SERVA IPG Strip equilibration buffer Eq buffer 3 Equilibrate each IPG strip gel side up in 6 ml solution in a SERVA equilibrator fig 5 2 on an orbital shaker with 30 rev min DTT solution Weigh urea and dithiothreitol DTT
3. DTT and add the equilibration buffer according to table 4 1 and dissolve completely IAA solution Weigh urea and iodoacetamide IAA and add the equilibration buffer according the table 4 1 and dis solve them completely Table 4 1 Preparing the equilibration solutions for 11 and 7 cm IPG strips Number x Size of Urea g DTT mg IAA mg Eq Buffer ml Total volume ml strips 1 8 50 5 6 2x11cm or 3x7cm 1 8 125 5 6 3 6 10 12 4x11cm or 6x7cm 3 6 250 10 12 5 4 15 18 6x11cm or 9x7cm j 375 15 18 8x11cm or 12x7cm 2 Equilibrate each strip gel side up in 3 ml 11 cm strips or 2 ml 7 cm strips solution in an equilibrator fig 4 1 on an orbital shaker with 30 rev min Step 1 in DTT solution for 15 min Step 2 in IAA solution for 15 min 3 After the 2nd equilibration discard the solution 4 Apply 45 ml of each electrode buffer to the respective electrode wick in the compartments of the SERVA PaperPool fig 4 2 distribute the solution evenly with a roller Fig 4 2 Fig 4 3 5 Remove excess electrode buffer from the wicks by tilting the electrode wicks along one long edge and dab it on the PaperPool bottom fig 4 3 13 6 Apply 3 ml of cooling contact fluid onto the cooling plate for good cooling contact 7 Switch the thermostatic circulator on set to 15 Switch the FlatTop Tower on and set the valve to By pass to avoid water condensation on the gel surface
4. ELECTRICAL REQUIREMENTS Mains 110 120 220 240 V AC 150VA max DC input 0 1500V DC 200mA max with 2mm to 2mm DC supplied cables only DC input 1 1000V DC 200mA max with 2mm to 4mm DC cable only accessory The supplied DC cables 2mm to 2mm connector type are rated for 1500V If cables or adaptors not supplied with the Tower are used ensure these have a suitable DC insulation compliance for the voltages used 5 OPERATING CONDITIONS Temperature 5 C to 40 C Altitude up to 2000m Relative Humidity up to 80 It is not recommended to operate the system under the influence of extreme electromagnetic interference such as in dustrial mains noise or extreme electrostatic discharges Operating under these conditions may cause the front panel indicator or the 20 current level indicator to show incorrect readings Should this occur it is recommended to iso late or prevent the external interference effect and if necessary cycle the power switch on and off to reset the proces sor It is recommended that the red and black leads from the power supply are twisted together to reduce possible RF emissions and to avoid tangling In all cases of interference the unit remains CE mark compliant CE CERTIFICATION This product meets the requirements of applicable CE directives IEC 61010 A copy of the corresponding Declaration of Conformity is available on request This CE approval is for the HPE FlatTop Tower only The operator is responsible
5. Users should identify and resolve problem before continuing with electrophoresis Check the set up power supply and ensure that only gels recommended for use on the FlatTop Tower are used Running less than 4 gels Between 1 and 4 gels can be run at any one time on the Tower To avoid alarms sounding the electrode lids of drawers that are not in use should remain unplugged 10 2 Loading and Running Gels In this section general instructions for loading and running gels on the Flat Top Tower are provided Running conditions for specific types of gel are described in subsequent sections video and other useful information on running a the HPE Tower is available for www serva de Equilibrating IPG strips SERVA IPG strip equilibrator provides a convenient way to equilibrate IPG strips The equilibrator has been designed so that strips can be conven iently transferred from one slot the first equilibration solution e g DTT to the second e g IAA Preparing Electrode Wicks Electrode wicks soaked in an appropriate buffer provide a convenient alter native to buffer tanks The wicks should be fully soaked for at least 15 min utes thus we recommend you prepare the wicks first For large format gels thicker wicks are used to provide sufficient buffer for a run Air in the wicks can be removed by applying gentle pressure using a roller before the stacks are applied onto the gel It is practical to soak the anodal and cath
6. for maintaining the regulatory approval for the additional devices attached to the HPE FlatTop Tower and CE mark ap proval for the system as a whole This includes the DC power supply DC cables and water cooler In order to maintain CE mark regulatory requirements if an alternate DC connecting cable is supplied by the user it must remain less than 3 metres in length Any alternate cables or adaptors used must have suitable insulation compliance The supplied DC cable 2mm to 2mm connector type is rated for 1500V The optional 2mm to 4mm cable type can only be used with DC voltages up to 1000V The CE symbol and corresponding declaration of conformity is valid for the instrument when it is e Used in indoors in a laboratory location Used with accessories described in this manual or recommended by SERVA Electrophoresis GmbH e Used in the same state as it was delivered from SERVA Electrophoresis GmbH except for alterations de scribed in this manual Packing List HPE FlatTop Tower comprising The Tower base unit 4 X lids packed separately containing the electrodes 1 X lamp for illumination of gels 1 mains cable 1 X 2mm power supply cables 1 X 1 m thick wall silicone tubing Handle for the bypass valve This manual HPE FlatTop Tower Accessories comprising e 4PaperPools e 1 small roller Please check the contents of your FlatTop Tower package and contact your local SERVA representative immediately if any items are missing I
7. 1W 2 W 3 W 4 W 30 min constant limit limit limit limit limit limit limit limit S2 200 V 13 mA 26 mA 39 mA 52 mA 3 W 6 W 9 W 12 W 30 min constant limit limit limit limit limit limit limit limit s3 300 V 20 mA 40 mA 60 mA 80 mA 5 W 10 W 15 W 20 W 10 min limit constant constant constant constant limit limit limit limit after this step remove the IPG strip S4 220 V 5 mA 10 mA 15 mA 20 mA 2 W 4 W 6 W 8 W Hours until limit constant constant constant constant limit limit limit limit 6 00 a m next day 1500 V 40 mA 80 mA 30 W 60 W 90 W 120 W limit constant constant constant constant constant constant constant constant Please note It is not recommended to run the gradient gel 2DGel flatbed 10 15 overnight The specifications constant and limit are added for programming the Bio Rad power pack 18 6 Staining of Flatbed Gel Fluorescent LavaPurple Staining Below is a protocol for LavaPurple staining optimised for film backed gels which longer steps than slab gels without backing because the solutions can enter the gel only from one side LavaPurple staining must be performed in a clean plastic tray We recommend against using trays that have been used previously for other stains e g Sypro Ruby Coomassie Transparent trays are OK Do not use metal trays Solutions and Buffers Solution 1 fixation and acidification Place 850 ml of high purity water into a 1L bottle then add the cont
8. 3D Gel itch P with iae switch reference icc Flat screen i er 006 Fig 11 Schematic drawing of the cable M lt X rail o Y or PET ScreenPicker box Benefits Space saving easy to store on lab bench dimensions length width height compared to huge automatic picker J Time saving accuracy easy to use no need for specific training no need for manual corrections of the gaps al ways encountered with automatic pickers Full workflow integration with generation of picking digester list Ready within seconds Costs saving no need for expensive instrument or maintenance contract e Error free picking with help of pick list and plate well identifier e Flexibility initialization file defining visual appearance and requirements of digestion robot like input file and re served positions on plate Reads any image file format or picking list Safety no transilluminator or dangerous UV sources e Security Linux avoids transfer of viruses by USB memory keys PC and Software protected 27 12 Trouble shooting Symptom 12 1 Effects during electrophoresis Green light on cool water control is not spinning but static red after set to Cooling Air bubbles between film backing and cooling plate Excess cooling fluid around the film support Water droplets on the gel surface No electric current drawer control lamps do not illuminat
9. IPG strip slot 2 3 mm above the line 19 matching 18 5 fig 5 5 Fig 5 5 Placing Flatbed Large gel on the cooling plate 16 8 Remove excess electrode buffer from the wicks by tilting the electrodes along one long edge and dab it on the Paper Pool bottom fig 5 6 Always hold the wick horizontally do not tilt it this would cause a higher buffer concentration on one side Ed Fig 5 6 Removal of excess buffer from the wicks Always hold the wick horizontally 9 Place the electrode wicks onto the gel edges overlapping the gel by at least 2mm Move the anodal wicks around the gel area to avoid dropping buffer on the gel surface fig 5 7 Fig 5 7 Applying the electrode wicks on gel edges 10 Trim the film support of the IPG strip on both sides Important The IPG strips should not be too dry Ensure that there is still a thin layer of buffer on the surface 11 Place the IPG strip gel side down anodal side to the right into the slot of the Flatbed Gel fig 5 8 Start in the middle to prevent accumulation of buffer towards one side Push it carefully towards the anode edge of the slot Slide along the backing of the strip with the forceps to ensure good contact to the bottom of the slot Fig 5 8 Application of the IPG strip into the slot of the 2DGel flatbed Always hold the strip horizontally 12 Apply 5 ISDS marker proteins to the marker well 13 Close the lid while lowering the electrodes on the
10. Na2CO0 0 03 formaldehyde 0 00075 Na thiosulphate about 3 min 6 25 g Na gt CO3 225 ul formaldehyde 94 ulNa thiosulfate 2 visual control Stopping 10 v v acetic acid 30 min Preserving 10 glycerol 250 ml 30 min Drying air dry on the support film When staining is done on a staining robot or the same day the second fixing step should be performed with a solution with the same composition like the Fixing 1 solution Do not fix over night in 40 ethanol this causes strong curling of the film backed gels add aldehydes shortly before use 8 4 Scanning of Visible Stained Gels Bio 5000 VIS Gel Scanner Cat No SERVA Bio 5000 White light scanner for transmission and reflection mode Linearity over 4 0 O D 22 9 Blotting and Blot Staining of Flatbed Gels SERVA Gel Remover Cat No HPE GRO1 For electrophoretic transfer of proteins on a blotting membrane the film backing must be removed from the gel The easiest and safest way to remove the film backing without damaging the gel layer is cutting with a thin wire The best way is using the SERVA GelRemover as described here blotting buffer containinc clamp san seil RR 20 methanol or IPA s p surface up Fig 9 1 Soak the gel for 10 min in Fig 9 2 Gel remover before loading Fig 9 3 After pre equilibration with blotting buffer containing methanol to the gel anodal buffer clamp the gel on the gel avoid stretching of the gel after re remover s
11. Orbital shakers if available provide the best results The gel may be left in solution 1 overnight with no negative effects Bring dye concentrate to room temperature and mix thoroughly prior to use Add LavaPurple concentrate to solution 2 before pouring onto the gel to prevent staining artifacts The staining solution must be made fresh not more than 30 minutes prior to use The staining solution can be reused once for staining within 2 hours provided it is still purple in colour The fixing solution step 2 can be re used for acidification step 5 Do not stain longer than 3 hrs as signal will decrease after this time If there is no time for scanning you can leave the gel in the acidifier solution 1 overnight LavaPurple stained gels can easily be post stained with semi colloidal Coomassie Blue staining or silver staining for manual spot picking See page 15 19 Sypro is a trademark of Invitrogen 7 Scanning of Backed Gels Fluorescent The SERVA Gel ScanFrame is designed to prevent the curling of plastic backed gels during imaging Itis particularly useful for the scanning of fluorescently stained or labelled gels as this can take a considerable length of time during which the gels may dry and begin to curl The ScanFrame comprises a set of 3 x 2 non fluorescent PVC frame bars to prevent curling of plastic backed gels dur ing scanning fig 7 1 Fig 7 1 The ScanFrame consist of two 10 x 25 x 265mm two 10 x 25 x
12. Staining similar sensitive like Coomassie Staining fully MS compatible SERVASnow Staining Kit Cat No 35080 Reversible staining method acc to Hardy et al Anal Biochem 240 1996 150 152 Limit of detection lt 10 ng Fast procedure about 20 min Subsequent blotting and elution of proteins is possible Prepare the working solution I Dilute Solution I 1 10 with ddH2O Prepare the working solution II Dilute Solution II 1 10 with ddH2O Wash the gel with ddH20 for 30 to 60 s after electrophoresis Incubate the gel 15 min in working solution I Remove the solution and rinse the gel with ddHzO Incubate the gel in working solution II until the background turns intense white Remove the solution and rinse the gel with ddH2O Incubate the gel 5 min in washing solution 1 50 dilution of SERVASnow Solution I with ddH20 O w No oS SS DN Seal the gel in new washing solution Store at room temperature Drained gels will become transparent After rehydration staining will be visible again Destaining and Mobilisation of Proteins for Blotting Prepare the mobilizing buffer 50 mmol L EDTA 25 mmol L Tris pH 8 3 0 76 g Tris 4 65 g EDTA Naz dissolve in 250 ml distilled water adjust to pH 8 3 with a few grains of Incubate the gel in the mobilizing buffer after staining The gel will become transparent again and blotting is possible 8 3 Mass Spectrometry compatible Silver Staining A number of different Silver Stai
13. at IPG strip s because of electroendosmotic effect 28 Remedy Check tubing Tubing leading from Qut of chiller must be connected to In on Tower Lift up gel on one side and apply a higher volume Remove excess fluid with lint free tis sue paper Apply smaller volume in future Set cool water flow to Bypass while setting up until the lid has been applied then switch cool water flow to Cooling Plug power supply cables in correctly black cable to cathode red cable to anode When less than four gels are run unplug the non used electrode lids and place them into the parking position Re position lides and check connecti ons between lids and drawers Switch cool water from Bypass to Cooling when starting electrophoresis Check chiller temperature and ensure no other aparatus is connected to the same chiller See above Relocate chiller or other apparatus If you run less than four gels at a time reduce the mA and W settings in the power supply accordingly Follow strict ly the manual Always hold electrode wicks horizontal when carrying them to the gel See section 1 of manual Remove IPG strip s from gel after the first 70 minutes and then continue the run Follow strictly the manual Symptom Minor disturbance s in the front Irregular bulging of the front on one side Run stops front does not continue to migrate sparking at the IPG strip s 11 2 Effects during sta
14. but do not switch on the FlatTop Tower at this point Connecting the Thermostatic circulator Chiller It is important to place the chiller in a position that avoids warm air exhausting from the chiller heating the Flat Top Tower In order to achieve optimal cooling effectiveness the chiller must be placed next to the Flat Top Tower on the same bench level The tubing connecting the Tower to the chiller should be as short as possible and insulated with foam With the bypass valve set to bypass connect the FlatTop Tower to the chiller using the tubing provided Fix the tubing using the hose clamps Jubilee clips provided It is important these clamps are sufficiently tight to provide an air tight seal The chiller Outlet must be connected to the Tower Inlet and the chiller Inle to the FlatTop Tower Outlet The flow direction must be correct otherwise the cooling system will not function Removal of Air For efficient cooling it is vital to remove any air from within the cooling plates in the Flat Top Tower After connecting the FlatTop Tower as described above switch on the chiller with the valve in the Bypass position Leave the chiller running until all of the air in the connecting pipes is removed this may take 2 3 minutes Move the valve position from Bypass to Cooling and leave the chiller running until little or no air is seen in the connecting pipes This will take around five minutes during whic
15. can be muted by pushing the Clear button Closing the drawer s should resolve the prob lem This alarm has been removed with the new version of the firmware Constant illumination of a green LED after the set up period indicates power is supplied to the corresponding drawer and that it is functioning correctly Orange warning light on a specific drawer indicates that power to that drawer is 20 greater or less than the average for all four drawers Power will continue to be supplied to the drawer and the alarm can be muted by pushing the clear button Check the set up of electrode wicks have been correctly positioned and correct buffers used Check and ensure good connection between elec trode and wicks Check that the gels suitable for the FlatTop Tower and the same gel types have been used in all drawers Check the correct and same number of electrode wicks applied to each gel Ensure that the cooling plate is properly cleaned Drawers can be run with the orange light illuminated but may show different migration rates Audible alarm plus red warning light indicates that the current to that drawer is gt 100mA Power will be shut down to ALL drawers and will not be reactivated until user intervention drawer opened power cycled clear button pushed This alarm will repeat if the issue has not been resolved The power shut down is to avoid excessive heating of the gel that could be hazardous and or result in damage to the equipment
16. not spill or store liquids on the top of the unit e If liquid is observed to have spilled or overflowed into the electronics in the top of the HPE FlatTop Tower immediately disconnect the high voltage power supply and the AC mains power to the entire instrument befo re opening the safety enclosure lid e Do not operate or connect power sources to the equipment if there is any mechanical damage e Do not obstruct access to the controls power switch and bypass valve they must remain freely accessible while operating WARNING THIS EQUIPMENT WEIGHTS 80KG e The instrument requires appropriate lifting tools to locate into position e Do not attempt to lift without appropriate occupational health and safety considerations e Do not open multiple draws at once The equipment may over balance and either equipment damage or hazardous conditions may result WARNING HIGH TEMPERATURE AND FIRE HAZARD e High temperatures can be generated at the gel surface Care should be taken when opening the drawer to asses the temperature before touching e Care should be taken not to allow the gel to dry out e Flammable gels must not be used e The equipment must be monitored by the operator at all times e Only use gels recommended for the HPE FlatTop Tower This warning symbol highlights instructions that must be followed to avoid personal injury It is impor tant not to continue until all stated conditions are met and clearly understood
17. suitable for the HPE FlatTop Tower 1D SDS PAGE 25 slots Marker muscle E coli extracts Coomassie staining 1D native PAGE 52 slots Tomato seeds Coomassie staining 1D SDS PAGE 103 slots Marker serum muscle seed proteins Coomassie staining 2D SDS PAGE running 3 IPG strips 7 cm Triple gel E coli extracts LavaPurple staining 2D SDSPAGE running 2 IPG strips 11 cm Double gels Human serum proteins LavaPurple staining 2D SDSPAGE 1 IPG strips 24 cm Large gels E coli proteins LavaPurple staining Coomassie is a Trademark of ICI corporation i er CleanGel 10 525 native Protein Electrophoresis tomato varieties EV EO NE an AN AHAI HHI iii i Important Safety Information PRIOR TO OPERATING THIS INSTRUMENT READ AND UNDERSTAND THIS MANUAL ENTIRELY THIS INSTRUMENT MUST BE USED ONLY BY INDIVIDUALS SKILLED IN ELECTROPHORESIS THIS INSTRUMENT AND ITS COMPONENTS ARE FOR IN VITRO RESEARCH USE ONLY WARNING OPERATION OF THIS INSTRUMENT REQUIRES HIGH VOLTAGE e Disconnect the high voltage external power supply before opening any drawers e Turn off and disconnect any high voltage power supply before opening the safety enclosure lid e Disconnect the high voltage external power supply and AC mains supply before opening top panel e Disconnect the high voltage external power supply and the AC mains supply before cleaning or servicing e Do
18. 190mm two 10 x 25 x 100mm PVC bars It is suitable of both SERVA large 25 x 19 5 cm and standard 25 x 12 5 cm format horizontal and vertical 2DGels SDSGels DNAGels and FocusGels These bars are placed on the edges of the gel to press the whole gel horizontally on the scanner platen fig 7 2 Standard Gel Format 2DGel flatbed Large Gel Format 2DGel vertical 2DGel flatbed Large Fig 7 2 The ScanFrame holding down a normal 25 x 12 5 cm and a large sized gel 25 x 19 5 cm The gels are scanned with the gel surface down facing the platen directly after applying a few ml water on it focal plane 0 or platen During scanning the ScanFrame is laid on the gel to avoid curling of the edges Do not apply Press Sample For accurate spot picking the scanning orientation must be flipped as shown here D are Important When you scan DIGE gels without fixing and or post staining pti ons sy Clean the platen with distilled water and dry it after each scan to avoid accumulation 0 anta Pi d ex of air bubbles originating from SDS buffer tg Mk Scanner Settings Excitation maxima 390 and 500nm Common excitation sources include UVA or 473 nm 488 nm and 532 nm lasers Emission maxima 610 nm Common filters 560 LP 610 BP30 600 BP10 Example using Typhoon 532 nm laser 540 PMT 610BP30 filter 100 um resolution normal sensitivity 20 Typhoon is a trademark of GE
19. 5 min complete de staining of the bands is achieved by incubating the film for 5 min in 0 2 mol L NaOH Ponceau S Staining Dissolve 0 1 w v Ponceau S in 1 v v acetic acid Stain for 5 min Destain two times for 5 minutes with 5 acetic acid 25 10 Pattern Evaluation and Image Analysis There are two types of software used in image analysis Algorithms of first generation detecting spots on all images independently followed by warping and matching before going to final results Algorithms of second generation aligning images prior to any spot detection followed by spot detection across series of gels followed by statistics and final results In both cases the quality of the gel image in terms of reproducibility and resolution is one of the most important factor that guarantee correct final results Recent reproducibility tests made on behalf of HUPO www fixingproteomics org showed that the reproducibility of high quality standardised electrophoresis gels was much higher that first expected It is common to see more than 90 of the differential soots common to all labs using second generation algorithms as described here above The combination of the HPE Flatbed Tower and HPE gels is ideal for high quality proteomics analysis where true differ ences induced by the disease or the treatment must be extracted from technical noise and biological variations Figure 10 1 shows the perfect auto alignment obtained on Second generati
20. Electrophoresis Heidelberg SPE TM Flatfop Tower User Manual HPE FlatTop Tower www serva de 1 Table of Contents HPE FlatTop Tower Examples of Gels Suitable for the Flatlop Tower Important Safety Information 1 2 8 9 Assembly and Installation Loading and Running Gels One Dimensional SDS Electrophoresis Two Dimensional Electrophoresis Double and Triple Gels Two Dimensional Electrophoresis Large Gels Staining Flatbed Gels Fluorescent Scanning Backed Gels Fluorescent Staining Flatbed Gels Colorimetric Blotting and Blot Staining of Flatbed Gels 10 Pattern Evaluation and Image Analysis 11 Band and Spot Picking from Flatbed Gels 12 Trouble Shooting Legal Information Ordering Information 11 12 15 19 20 21 23 26 2 28 30 30 HPE Flatlop Tower The HPE M FlatTop Tower is used for one dimensional and two dimensional electrophoresis gels where multiple con sistent runs are required The unit consists of four horizontal electrophoresis chambers which are built as drawers into a metal housing The instrument is run with an external power supply and thermostatic circulator chiller The gels are protected from light during the run which prevents bleaching of fluorescent labels No cassettes are re quired as the plastic backed gels are run directly on an alumina ceramics cooling plates The specially formulated HPE ge
21. Healthcare 8 Staining of Flatbed Gels Visible Detection 8 1 Colloidal Coomassie Staining SERVA HPE Coomassie Staining Kit Cat No 43396 SERVA HPE Coomassie Staining Kit is a highly sensitive staining method for 1D and 2D gels after electrophoresis on based on the colloidal Coomassie Blue G acc to Neuhoff et al Electrophoresis 9 1988 255 262 that uses dist water as a destain All reagents are MS compatible 1 Fix gels 2 x 1 h with 15 Ethanol 1 citric acid 2 Wash the gel for about 5 min in distilled H2O before staining 3 Mix the dye solution containing 98 v v Solution A 2 v v Solution B for several hours The final stain ing solution contains 80 v v dye solution and 20 v v ethanol For a large 2D gel the volume of the staining solution should be at least 300 ml Stain gel with gentle shaking shaker with ca 50 100 rpm for at least 3 hours Staining over night is optimal The staining time will be reduced by half when staining is performed at 40 C to 45 Destain Wash gel after staining in dist H2O with regular exchange of the H2O The band intensity will increase significantly after neutralization in water Note Coomassie staining can also be used as a fixing procedure prior to silver staining After de staining the back ground continue directly with the sensitizer step SERVA ScanFrame is also useful to prevent curling during the imaging of Coomassie stained gels 8 2 Negative
22. and add the equilibration buffer according to table 5 1 and dissolve completely IAA solution Weigh urea and iodoacetamide IAA and add the equilibration buffer according the table 5 1 and dis solve them completely After the 2nd equilibration step discard the solutions Table 5 1 Preparing the equilibration solutions for 24 cm IPG strips Number of strips Urea g DTT mg IAA mg Eq Buffer ml Total volume ml Step 1 in DTT solution for 15 min Step 2 in IAA solution for 15 min 15 fim i Fig 5 2 Equilibration of the IPG strips in 6 ml equilibration buffer 3 Switch the thermostatic circulator on set it to 15 C Switch the FlatTop Tower on and set the valve to Bypass to avoid water condensation on the gel surfaces 4 Apply 4ml of cooling contact fluid onto the center of the cooling plate fig 5 3 Fig 5 3 Application of the cooling fluid 5 Grip the gel surface up at the two lateral edges at the protruding film bend into a U shape and slide the film backing left and right to the lateral sides of the cooling plate to distribute the cool contact fluid evenly Fig 5 4 Fig 5 4 Placing the gel on the cooling plate distribute the cooling fluid evenly 6 Remove excess cooling fluid along the film edges with lint free paper tissue 7 Finally lower the the gel onto the cooling plate the IPG strip slot towards the cathode the cathodal edge of the
23. arge sized blots use 200 ml volumes Basification Wash the blot in solution 2 for 10 minutes Staining For small blots add 250 ul LavaPurple to 50 ml of high purity water For large blots add 1 ml of LavaPur ple to 200 ml of high purity water Stain in LavaPurple for 15 30 min Blots should be placed protein side down in the prepared stain Follow either the PVDF or nitrocellulose protocol from this point PVDF Acidification Place the blot in Solution 1 and rock gently for 5min For large blots use 400ml For small blots use 50ml This treatment will cause the blot to appear green Washing Rinse blot with 100 methanol for 2 3 min until green background on blot has been completely re moved Multiple rinses may be required Drying Dry for 2 3 min To allow simultaneous drying of the blot on both sides it may be best to dry the blot ona wire mesh Allow the blot to completely dry The blot is then ready for imaging and further analysis Nitrocellulose Washing Place the blot in Solution 2 and rock gently for 5min Remove from Solution 2 and place into high purity water and rock gently for 5min Repeat the water washing Allow blot to completely dry Your blot is now ready for imaging and further analysis Care should be taken to ensure that the membrane does not dry during the staining Fast Green staining Dissolve 0 1 w v of Fast Green in 1 acetic acid Stain for 5 min de stain the background with distilled water for
24. buffer and apply the stack onto the gel 8 Apply the cathodal electrode plate and start the electro transfer no cooling Example Apply 200 mA for a Flatbed gel 25 x 12 5 cm om poe e 24 Staining of the blot membranes LavaPurple LavaPurple provides the most sensitive method for the staining of protein blots which due to its reversible binding mechanism it is fully compatible with downstream processing such as Mass Spec immuno or functional staining Full details of blot staining with LavaPurple along with other useful information on blotting can be found at in section 6 of this manual Solutions and Buffers Solution 1 fixation and acidification Place 850 ml of high purity water into a 1L bottle then add the contents of packet A and mix until dissolved Add 150 ml of 100 ethanol and mix thoroughly Solution 2 staining buffer To prepare the staining buffer add 1L of high purity water to a 1L bottle and add the 6 2 g H3BO3 Dissolve the powder by mixing using a magnetic stirrer may take several minutes then add 3 84 g NaOH and continue stirring until fully dissolved Solution 3 washing Mix 850 ml of high purity water and 150 ml of 100 ethanol in a 1 L bottle Storage of solutions Solutions 1 3 can be stored at room temperature and are stable for up to 6 months Washing Following transfer place the wet membrane in water and wash for 3 x 5 min For small sized blots use 50 ml volumes for all steps For l
25. e after starting the power supply Condensation inside of electrode lid Front is curved instead of straight Migration of front is very slow and will not reach the anode in time Front is slanted not straight Audible alarms and lamps flashes orange or red Condensation water develops inside electrode lid near to IPG strip s Cause Tubing connection to the chiller is wrongly connected or kinked Volume of cool contact fluid was not sufficient amount fluid required de pends on ambient temperature Too much cooling fluid applied on the cooling plate amount fluid required is depends on ambient temperature Gel is pre cooled without lid at high humidity conditions leading to water condensation Electronic control detects wrong orien tation of electric field Electronic control detects that one or more draws do not contain a gel Lids not properly postioned or not plug ged in The gel gets hot during electrophoresis because of insufficient heat dissipation The gel gets hot during electrophoresis because of insufficient heat dissipation FlatTop Tower is subject to hot ex haust from chiller or other apparatus The gel gets hot during electrophoresis because too much power is appied per gel The electric field is too low Uneven buffer concentration within the electrode wicks Not all gels and wicks are connected leading to differences in current beteen the gels Local heat production
26. electrode lids in the lid carriers contained in the 3 drawer above Never put the lids electrode side down on the bench as this may damage the plati num wire Clean the platinum electrodes with a lint free tissue moistened with distilled water Rub the tissue only up and down the electrode never across the electrodes The electrode positions can be adjusted to two different gels sizes standard and large size When changing the positions leave the electrode lid in the parking position to prevent damage to the electrodes Be careful to catch the nut on the underside of the lid into which the screw fits Warning lights and alarms Green power light mains power is connected and switched on Green rotating LEDs indicate water is flowing through the cooling plates Red stationary LEDs water is not flowing through the cooling plates Power supply alarms The current flow in individual drawers in the Tower are monitored and different alarms indicate different problems To facilitate monitoring there is a 2 3 minute delay between switching on the power supply and constant illumination of the individual drawer lights During this 2 3 minute delay period the corre sponding LED in any drawer where a current is detected will flash green Audible alarm alone that beeps every 4 seconds indicates that one or more drawers have been open whilst the external power supply was on The LED on open drawers will not be illuminated Alarm
27. entle rolling Fig 3 2 6 Pipette 15 ul 25 S or 6ul 52 S of sample into the sample wells 7 Clean platinum electrode wires before and after each electrophoresis run with moist tissue paper 8 Close the lid while lowering the electrodes on the wicks plug the cables in turn the valve to Cooling 15 C Table 3 1 Running conditions 15 C Quick run for normal samples total 2h Steps Voltage Current Power Time r zots 3 els ages 1901 2gels sgets ages S1 600 V 42 i 84 H E mA E mA 30W 60 W 90 W 120 W 1h TT Ti Ti a TT 1000 V 50mA 100 mA 150 mA 200 mA er W aan W a W Er tf tt tt vi tt Table 3 2 Running conditions 15 C Slow run for difficult samples total about 2 h 30 min S1 250V 30mA 60mA 90mA 120mA 10w 20W 30w 40 W 45 min Tt i f i f o m tt tt S2 700V 42mA 84mA 126mA 168mA 30w 60W SOW 120W 45 min tt ih ial tt o i f f S3 1000 V 50mA 100mA 150mA 200mA 50W 100W 150W 200W 1h t o Ti mi TT f f f f For programming BioRad Power Supplies only Set as constant tr Set as limit 12 4 Two Dimensional Electrophoresis Double and Triple Gels Always wear powder free disposable gloves Important Only use the SERVA buffer kit for the running buffers and equilibration solutions 1 Prepare the two equilibration solutions from the SERVA IPG Strip equilibration buffer Eq buffer DTT solution Weigh urea and dithiothreitol
28. ents of packet A 10 g citric acid and mix until dissolved Add 150 ml of 100 ethanol and mix thoroughly Solution 2 staining buffer To prepare the staining buffer add 1L of high purity water to a 1L bottle and add the 6 2 g H3BO3 Dissolve the powder by mixing using a magnetic stirrer may take several minutes then add 3 84 g NaOH and continue stirring until fully dissolved Solution 3 washing Mix 850 ml of high purity water and 150 ml of 100 ethanol in a 1L bottle Solutions 1 3 can be stored at room temperature and are stable for up to 6 months Table 6 1 LavaPurple staining of 0 65 mm thick film backed polyacrylamde gels Standard Large lenene fixing gem 1 300 ml 400 ml 30 min Dispose of this solution after use 2 Fixing Solution 1 30 min Solution 1 from this step may be reused for 300 ml 400 ml to over acidification step 5 night 3 Staining 0 5 v v LavaPurple in Solution 2 300 mi 400 mi 1 5 ml Lava 2 0 ml Lava Purple Purple 4 Washing Solution 3 300 ml 400 ml 30 min Solution 3 may be re used up to 2 X 5 Acidify 30 min Solution 1 300 ml 400 ml to over night Notes The gels must be completely covered with the various solutions Staining plastic trays should be selected that are only marginally larger than the gels and gels should not stick to the bottom during staining Gels should be placed gel side down in the staining solution Minimize carry over of solutions between steps
29. er which can result in uneven contact Electrodes should be cleaned with a lint free tissue moistened with dis tilled water If the electrodes become tarnished they should be cleaned with a silver cleaning cloth then wiped clean using a lint free tissue mois tened with distilled water To minimize contamination e g keratin wear powder free gloves face masks lab coat and a hair net Handle wicks using bunt end and IPG strips using pointed end forceps For fluorescence labeling and staining use lint free tissues and low fluorescence materials Switching on the Tower The FlatTop Tower has a internal pump that must not be run dry To avoid this always switch on the chiller first with the valve in the Bypass position Allow the chiller to pump for 2 3 minutes with the valve in Bypass to ensure no air from the chiller is pumped into the cooling plates When no air is apparent in the chiller tubing switch on Tower mains at the mains The left green light indicates that the main power is on the LED ring will show stationary red until the bypass valve is open If there is an audible alarm ensure all drawers are closed and push the the red Clear button The bypass function In horizontal electrophoresis gels are applied directly onto the cooling plate If the gels are cooled before the lid is applied this can lead to water condensation on their surface Therefore before and during loading the gels the bypass valve should be s
30. et to Bypass After gels IPG strips electrode wicks and electrode lids have been applied the valve must be changed to Cooling Ensure that the bypass valve is fully open when running gels by pulling it firmly to the cooling posi tion If the valve is not fully open gels may not be effectively cooled 8 Positioning the Gel With the Flat Top Tower backed gels are applied directly onto a ceramic cooling plate To facilitate heat transfer from the gels during electrophoresis cooling fluid included in buffer kit is applied to the surface of the ceramic plate before the gel is applied Applying the lids The lids of the FlatTop Tower play an important role as they house the electrodes that contact the buffer wicks When not in use insert the lids into their park positions below the drawers with the connecting plugs inside the FlatTop Tower Switching off the FlatTop Tower Switching off the FlatTop Tower should be done in the reverse sequence Used wicks can be disposed of into normal waste The cooling plate and electrodes should be immediately cleaned using a distilled water and a lint free tissues Do not leave gels wicks or cooling fluid on the cool ing plates as this can result in residues that are more difficult to remove Care with the electrodes and changing electrode positions The electrodes are carefully constructed from platinum wire and can be easily damaged if not handled correctly To avoid damage store the
31. f the gel is below 7 Sample pre treatment Double the sample volume by adding an equal volume of sample buffer 2x then dilute the sample to acheive the an appropriate gel loading concentration this depends on the sensitivity of staining method used e g Coomassie Blue Silver Staining or LavaPurple using 1 x sample diluter Then reduce and alkylate your sample 1 Switch the thermostatic circulator on set to 15 C Switch the FlatTop Tower on and set the valve to By pass to avoid water condensation on the gel surface 2 Lay two electrode wicks into the compartments of the Paper Pool Add 45 ml of the respective electrode buffer to each wick distribute the solution evenly with a roller and allow to soak for at least 10 minutes Fig 3 1 EA 3 Apply 3 ml cooling contact fluid onto the cooling plate 4 Remove the gel from its packaging Remove the cover film Grip the gel surface up at the two lateral edges at the protruding film bend it like an U and slide the film backing left and right on the cooling plate to distribute the cool contact fluid evenly Fig 3 2 Lay the gel down on the cooling plate being careful to ensure no air is trapped behind the gel Remove excess cooling fluid along film edges with lint free tissue paper 5 Place the cathode strip onto the cathodal edge of the gel The edge of the strip should overlap the gel not more than by 2 mm Place the anode strip on the anodal edge and remove any air by g
32. for soaking the electrode strips 4 HPE A02 IPG Strip Equilibrator Tray for IPG strip rehydration and equilibration 1 HPE A04 ScanFrame for standard and large size gels 1 HPE A05 Cooling fluid Cooling Contact Fluid 50 ml 1 43371 01 150 mi 1 43370 01 For IPG strips Gels and Buffer kits see SERVA HPE Catalogue Additives Servalyte Mix 2ml 40 w v for rehydration of IPG strips 1 pack 42940 04 Urea 1 kg 24524 02 CHAPS 59 17038 02 Dithiothreitol DTT 5g 20710 03 lodoacetamide IAA 5g 26720 01 SDS marker proteins examples only for other standards see www serva de Dual Color Protein Standard III 7 240 kDa 500ul 39252 01 Recombinant SDS PAGE Protein Marker 10 150 KDa PLUS liquid mix 500ul 39218 01 Staining LavaPurple Fluorescent stain 25 ml 43373 01 4x 25 ml 43373 02 HPE Silver Staining Kit 1 kit 43395 01 HPE Coomassie Staining Kit 1 kit 43396 01 EUROPE SERVA Electrophoresis GmbH Carl Benz Str 7 D 69115 Heidelberg Germany Phone 49 6221 13840 0 Fax 49 6221 13840 10 E Mail info serva de SERVA 30 Electrophoresis
33. g and the black cable to the black plug When the cables are plugged in the wrong way the electronic control in the FlatTop Tower will detect this and automatically disconnect the electric cur rent For the HPE Power Supply 1500 you will need to insert the adaptor provided into the end of the lead The supplied DC cables 2mm to 2mm connector type are rated for 1500V If cables or adaptors not supplied with the Tower are used en sure these have a suitable DC insulation compliance for the voltages used When running the Tower do not open a drawer until you have switched off frozen paused standby Wait the power supply Opening the drawers with the power supply running causes your power supply to detect a ground leakage which results often in a disturbance of the running pro gramme Cleaning To obtain the best results with the FlatTop Tower requires that it is scrupulously clean and we recommend cleaning before and immediately after use When first running the FlatTop Tower or if the cooling plates become soiled they should be cleaned using a 0 1 SDS solution followed by isopropyl alcohol and finally distilled water Subsequent cleaning can involve cleaning with distilled water alone The cooling plate can be gently rubbed and dried using a lint free tissue Abrasive cleaners and other solvents must not be used Cleaning of the platinum electrodes after each run is particularly important to prevent crystallization of buff
34. h time you will need to refill the chiller reservoir as the FlatTop Tower takes up about 2 litres of water With cooling water through the system switch on the FlatTop Tower The green light indicates that the power is on the internal pump will be heard to start The green H20 LED ring will illumi nate and rotate approximately 2 rotations per second If the red stationary LEDs are seen or the green LED rotates slowly something is wrong Most likely this will indicate that the tubing from the chiller is connected back to front is kinked or the valve is in the Bypass position An alarm will sound if any drawers are open this can be muted using the clear button and by pushing the drawer back into position To remove the last traces of air push the valve between Cooling and Bypass creating a hammering effect that should remove any last traces or air Watch the return tubing to the chiller to determine when the air has been fully removed Leave running for 30 mins to ensure all traces of air have been removed Electrode lids Take the electrode lids out of the package Remove the protection film and paper Insert the lids into their park positions below the drawers with the connecting plugs inside the FlatTop Tower Connecting the power supply The power supply is connected with the FlatTop Tower using the black and the red cable sup plied do not mix these cables connect the red cable to the red plu
35. he gel rather than over the top of the gel Applying the IPG strip The plastic film support on both sides of the IPG strip must be trimmed just beyond the gel The strip should be carried horizontally and applied to the slot centre first The strip should be placed in the IPG slot gel side down with the anodal side to the right To ensure good contact in the slot the back of the forceps are slid gently along the back of the IPG strip Applying the lid The lid containing the electrodes is slid from its parking position carefully placed over the gel and plugged in at the back of the drawer The valve set to cooling and the power supply switched on The power supply MUST be switched off or to standby freeze paused before the drawers are opened 11 3 One Dimensional SDS Electrophoresis 1D SDS PAGE kits contains ready to use SDS polyacrylamide gels running buffers paper wicks and sample diluters are available from SERVA The gels are polymerized on plastic backing have a size of 25 x 11 5 cm x 0 45 mm thick and are available with either 25 x 15 uL slots 25S x or 52 slots x 6 uL 52 S slots Various gel concentrations are available 10 T 12 5 T 15 T The gels are backed using a non fluorescent film specifically designed for fluores cent pre labelling of proteins DIGE and or fluorescent staining LavaPurple For long shelf life and optimal separation a Tris tricine gel chemistry is used which maintains the pH o
36. ining For all staining methods Gel comes off the film support LavaPurple staining Dye turns from light purple to light yel low 12 3 Effects during scanning Gel edges curl up during scanning 12 4 Effects seen in the result Horizontal streaking Vertical streaking Local disturbances in the pattern LavaPurple staining Unsufficient sensitivity of staining Cause Buffer drop s fell on the gel surface Air in between wick individual wicks Equilibration buffer unequally distribu ted within the IPG strip s Strong electroendosmosis effect at the IPG strip s because it has not been removed after the first 70 minutes Too high alcohol concentration for film backed gels Staining solution too acidic Volume of staining solution too low Gel edges start to dry out The first dimension IEF separation in the IPG strip did not work well because of inappropriate sample preparation IEF separation problems Insufficient equilibration of the IPG strip IPG strip has become too dry Air bubble in a buffer wick Wrong protocol for non backed gels has been applied or the solution volu mes were too small 29 Remedy Avoid passing wicks over gel surface Gently roll wicks in Paperpool to remo ve alr Hold the IPG strip s horizontal start in the middle when placing the strip into the slot Remove IPG strip s from gel after the first 70 minutes and then continue the run Follow st
37. llows performing a fine tuning between the size of the displayed image with a mouse and the size of the real gel with the help of markers or punched holes used as landmarks An XY carriage is used to guide the manual picker over the target to be excised and to prevent parallax errors The ScreenPicker guides visually the user to place the picked plug in the right well of the microtiter plate using the file generated by the evaluation software At the end of the process a file containing spot numbers and microtiter plate plug positions is generated for use by digester robot Because it clearly marks the spots to pick on the image the ScreenPicker can also be used for Coomassie blue or silver stained gels increasing the picking accuracy and making the process entirely error free Another interesting application is the picking of protein spots from 2D gels after the immunological visualization of spe cific spots by western blotting The scanned image of the blot membrane is projected under the gel which contains still a great portion of the proteins after the electro transfer The files from the evaluation software are transferred to the Netbook PC via an USB stick The special Crunchbang Debian Linux installation on the Netbook PC is minimalistic The Netbook PC setup is not designed for any other pur pose than picking spots One touch 2D gel spot picker I Power if cord ee 11 2D Image _ ere Mains
38. ls allow high voltages to be used in the electrophoresis and when combined with the very thin gels 0 65mm results in highly focused electrophoretic spots and bands The specially designed cooling plates and inbuilt pump ensu res efficient heat dissipation thus rapid and straight electrophoretic migration An electronic sensor system delivers in formation about the electric field distribution between the gels and indicates which drawer chambers are in use The system does not use buffer chambers instead wicks are soaked with concentrated electrophoresis buffers and placed between the gel edges and the electrodes that are mounted into the lids The electrode positions are adjustable to ac commodate two different gel sizes HPE M gels are available in two different sizes Standard size 25 x 11 5 cm for e 1D SDS or native electrophoresis with 25 52 and 103 sample wells e 2D electrophoresis accomodating 2 x 11 cm IPG strips and 1 MW standard lane e 2D electrophoresis accomodating 3 x 7 cm IPG strips and 2 MW standard lanes Large size 26 x 20 5 cm for e 2D electrophoresis with one 24 cm IPG strip and 1 MW standard lane Water circulation Input from Alarm Clear LED ring Power Supply button gelcompany t l z Outlet to chiller Inlet from chiller Bypass valve Plastic backed HPE gels on ce ramic cooling plate Anode buffer Drawer LEDs Wick Cathode buffer wick 3 Examples of types of HPE gels
39. n 20 methanol buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose Fig 9 10 Example for a setup for se midry blotting with a discontinuous buf 23 fer system semi dry blotting with a discontinuous buffer system Transfer buffers Anode solution I 0 3 mol L Tris 36 39 20 v v methanol 200 ml make up to 1 L with distilled water Anode solution Il 25 mmol L Tris 3 03 9 20 v v methanol 200 ml make up to 1 L with distilled water Cathode solution 40 mmol L aminocaproic acid 5 2g 0 01 w v SDS 0 19 20 v v methanol 200 ml make up to 1L with distilled water Methanol can be replaced by isopropanol IPA Blotting procedure with discontinuous buffer in a semidry blotter 1 For best results run the buffer front off the gel during electrophoresis prior to transfer 2 Wet the graphite anode plate the plate with the red cable with distilled water remove the excess water with tissue paper 3 Cut the necessary filter papers 6 for the anode I 3 for anode II 9 for the cathode and the blotting membrane to the size of the gel 4 Slowly soak 6 filter papers in anode buffer and place the stack in the middle of the blotting plate 5 Slowly soak 3 filter papers in anode buffer II and place the stack on top of the first stack 6 Remove the gel from the film backing see previous page and place it together with blot membrane on the stack Te Slowly soak 9 filter papers in cathode
40. n addtion to the FlatTop Tower you will need a suitable Power Supply and Chiller which are available from SERVA Electrophoresis GmbH If you have any comments on this manual please send them to us at SERVA Electrophoresis GmbH Carl Benz Str 7 D 69115 Heidelberg Germany Falcon is a Trademark of Becton Dickinson 6 1 Assembly Installation and Running Unpacking and Assembling of the HPE FlatTop Tower The HPE FlatTop Tower is transported in protective packaging The base unit and the 4 electrode lids are packed separately and require assembly at the final destination place A mechanical lifting device and at least 2 people should be used to lift the FlatTop Tower A ShockWatch label is included on the outside of the packaging which should be intact on arrival Please inspect this and report if the ShockWatch labels show damage or are missing to your local SERVA representative The Base Unit Place the base unit on the lab bench but do not plug in the power cable at this stage With a spirit level carefully level the base unit using the adjustable feet Remove the bypass handle and lamp from the accessories package Screw on the bypass valve handle Fix the lamp to the compartment lid using the two screws provided Open the lid by removing the 2 screws and connect the lamp cable inside the compartment Replace the lid screws Connecting the main power cable Plug in the supplied power cable 220V 110V at the back of the base unit
41. ning methods are available The two methods below are optimised due for MS compatibility for staining of backed gels Note Film backed gels need more and longer steps than slab gels without backing because the solutions can diffuse into the gel only from one side 8 3 1 Silver Staining Kit SERVA HPE Silver Staining Kit Cat No 43395 Sensitivity of detection about 0 1 ng BSA You can stain the gels also with the following lab made silver staining procedure 8 3 2 Lab made Silver Staining Table 8 1 MS compatible Silver staining of 0 65 mm thin film backed polyacrylamide gels step reagent volume time Fixing 1 10 acetic acid 15 ethanol 1 citric acid 250 ml 30 min 25 ml acetic acid 100 ml ethanol 2 5 g citric acid Fixing 2 10 acetic acid 15 ethanol 1 citric acid 250 ml Overnight 25 ml acetic acid 37 ml ethanol 2 5 g citric acid 4 x Washing H2O dist 4 x 250 ml 4x 5 min If the gel has been prestained with LavaPurple or semicolloidal Coomassie Staining fixing and washing is not necessary start directly with the sensitizing step Sensitizing 0 025 w v Na thiosulphate 10 mM Na acetate 30 ethanol 250 ml 40 min 63 mg Na thiosulfate 350 mg Na acetate 75 mIEtOH 4 x Washing H20 dist 4 x 5 min Silvering 0 2 AgNO 0 03 formaldehyde w v 40 min 500 mg AgNO3 225 ulformaldehyde 37 3 x Washing H20 gist 3 x 250 ml 3 x1 min Developing 2 5
42. odal wicks in separate PaperPool rather than side by side in a single pool to avoid cross contamination between the buffers The wicks should be moist but not dripping and sufficient buffer should be added so there is a small amount of buffer remaining in then SERVA Paper Pool The wicks should be rolled to remove airbubbles and to distribute the buffer evenly Remove excess electrode buffer from the wicks by tilting the electrode wicks along one long edge and dab it on the paper pool bottom When moving the wicks always hold them horizontal as holding them at a vertical angle can result in unequal buffer concentration Loading a gel Place the valve is in the Bypass position to ensure that the gel is not fluid is added to the surface of the cooling plate This is then dispersed by sliding a gel from side to side using the gel bent into a U shape The gel is amp then gently lowered avoiding air bubbles between the cooling plate and gel The grid lines on the cooling plate are used to correctly locate the gel Ex cess cooling fluid from around the gel is removed using a lint free tissue The electrode wicks are then applied with the cathode white at the front anode blue at the back The electrode wicks should overlap the gel by at least 2mm It is important that buffer is not dropped onto the gel surface and you should avoid moving the buffer soaked wicks over the gel For the an odal wick take the wick around the back of t
43. on software algorithms SameSpots Nonlin ear Dynamics Fig 10 1 ameSpots Auto alignment of 2 Lava Purple stained gels with 50 ug E coli and 2 Coomassie stained gels loaded with 1 mg E coli The 4 gels were HPE Flatbed NF 10 15 run together on HPE Flatbed Tower Such a high alignment quality is a must in Proteomics This can only be obtained by combining reproducible sample preparation reproducible gels standardised first and second dimension migration and to finish the use of a software algorithm that do not create additional variations like a variable number of spots between gels 26 11 Band and Spot Picking SERVA HPE ScreenPicker Cat No HPE SP1 Semi manual spot picking system The SERVA HPE ScreenPicker is a device to semi manually pick fluorescent 2D gels DIGE LavaPurple Sypro Ruby Flamingo without the need of post staining and or sophisticated robot and software It consists of an embed ded screen connected to a small computer to display the scanned image of the gel an XY carriage to guide the picker accurately over the spot position and a software which can read images and picking lists from most evaluation pack ages The ScreenPicker is adapted for gels on 3 mm glass plates fluorescent markers placed on 3 corners before scanning and HPE gels on non fluorescent plastic backing punched holes performed on 3 corners before scan ning The scanned image is projected under the gel The provided software a
44. rictly the manual Reduce ethanol concentration to 15 v v Film backed gels do not require high alcohol concentrations for fixing poteins because the gels cannot swell Apply the two pre buffering steps pro posed for film backed gels Follow strictly the manual for film backed gels Stain gel in the proposed volume Apply Gel Scan Frame on the edges of the gel during scanning Check the trouble shooting guides supplied by the providers of the IPG strips and web forums Use the equilibration buffer supplied with the HPE gels weigh out the cor rect amounts of DTT and IAA should be of highest reagent quality follow the manual Ensure that there is still a thin layer of buffer on the surface Distribute the buffer solutions evenly in the wicks by thoroughly rolling Follow the protocol of this HPE Tower manual which has been optimised for the HPE Flatbed gels Legal Information All goods and services are sold subject to the terms and conditions of sale of the company within gelcompany which supplies them A copy of these terms and conditions is available on request SERVA Electrophoresis GmbH Carl Benz Str 7 D 69115 Heidelberg Germany Ordering Information Quantity SERVA code no Equipment HPE FlatTop Tower 1 HPE TO1 HPE Thermostatic Circulator 1 HPE CU1 HPE Power supply 1 HPE PS1 Gel Remover 1 HPE GRO1 Bio 5000 VIS Gel Scanner 1 Bio 5000 HPE ScreenPicker 1 HPE SP1 Accessories PaperPool Tray
45. urface with the film backing moval from film down film backing with cut off gel _ Fig 9 4 Grip the the cutting wire and Fig 9 5 Grip the two handles and pull Fig 9 6 Remove the cutting wire and MT the wire between gel and film backing take off the gel with the film backing hitch it to the right handle in one smooth movement dry PVDF or s A o j film backing l blot membrane lt lt a with gel z re NC membrane i for PVDF membrane methanol for IPA bath Don mm Fig 9 7 Place film backing and gel on Fig 9 8 Flip the gel so the backing is Fig 9 9 Grip blot membrane and gel a flat bench and apply a dry blotting uppermost and remove the plastic at two corners and place them on the membrane to the gel Do not try to backing from one corner holding the filter paper stack correct any fold of the gel at this stage gel corner down with a spatula For as you will damage the membrane PVDF membranes immerse membra ne and gel in methanol or isopropa nol for a minute and smooth out any folds Other blotting transfer systems can be used for example tank blotting or semidry blotting with a continuous buffer system However the discontinuous buffer system described here is the most efficient 1 blot men gt Eiet Methanol can be replaced by isopropanol IPA SAM fik vane 1 Tovey ER Baldo BA Comparison of semi dry and conventional tank all buffers contai
46. wicks turn the valve to cooling 15 C switch on the FlatTop Tower main switch pumps and electronic control and start the run according to tables 2 or 3 see below 14 After 1 hour 10 min interrupt the run by standby pausing press wait freezing the power supply opening the draw ers and remove the IPG strip s and continue the run Tab 5 2 Running conditions day run cooling temperature 15 C Steps Voltage Current Power ter 2gele se soes ger 2905 gele 2925 S1 100 V mA Ed ka kog mA 1 W 2 W 3 W 30 min constant Fe Ed ka kog limit limit limit il 200 V 13 mA 26 mA 39 mA 52 mA 3W 6W 9 W 12 W 30 min constant limit limit limit limit limit limit limit limit 300 V 20mA 40mA 60 mA 80 mA 5 W 10 W 15 W 20 W 10 min limit constant constant constant constant limit limit limit limit after this step remove the IPG strip 1500 V 40 mA 80 mA 120 mA 160 mA 30 W 60 W 90 W 120 W 3h 50min limit constant constant constant constant limit limit limit limit 1500 V 45 mA 90 mA 135 mA 180 mA 40 W 80 W 120 W 160 W 50 min limit constant constant constant constant limit limit limit limit valid for homogeneous gels for the gradient gel 10 15 this step 4 takes 4 h 50 min Tab 5 3 Running conditions over night for homogeneous gels cooling temperature 15 C Steps Voltage Current Power Time roe 2gels seis eis 1901 2gels sgets ages S1 100 V 7MA 14mA 21mA 28 mA
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