E-PAGE 96 Protein Electrophoresis System
Contents
1. 3 Invitrogen life technologies Instruction Manual E PAGE 96 Protein Electrophoresis System For high throughput electrophoresis of proteins Catalog nos EP096 06 EPST96 06 Version C 27 January 2005 25 0668 ii Table of Contents Tabl Of Contents sensa a ps sanean EEEE dilantin gnc iii E PAGE Experienced Users Procedure sssssssssssussrssrassersersssensersees v Contents and Stoti genier re a sate E eE EE EES vii Specifica Hon Siagi a o a E beaded obese E e viii Introduction sesssssssnsnnsnneensnnnnnennnnnnnnnnnnnnnnnnnnnnnnunnnennnenennnnnenn enema 1 COVEL VIC W AE EE T 1 MethodS sisncessecsctnsecsssattenacesnorsteent coeds theta tednctlshastiiensehacestecstiacethedesues 4 Sample Prepatation sinises eiie ee aape E E Ee a T EE 4 Electrophoresis of E PAGE 96 Gels sssinssdenienamaiitivaenadationnaniade 13 Visualizing Lumio Fusion Proteins sssssesiisssseeitrrsseseettrrsssseerrsrssereer 16 Opening the E PAGE 96 Cassette 2 scucctsiseicsnuiictsicheitenescteisetentohed 17 Coomassie R 250 Staining Protocol ssccssecseeseseeeeesneeeesneeessneeesen 18 Drying E PAGE 96 Gels ssssssiissssesttirsssetttsrssesttrtsssststtrrssseeterrssstererrst 20 Semi Dry Blotting of E PAGE 96 Gels s sssssessssessesssssssessensesessensens 21 Using the E Holder Pla thorn ycsssiet3ep0i5 35 asuensaunt tend ane Gaus Weckewsesusid 24 Expected Results scccs scsesssssescsecdeabecsents2. The digital display shows EP or last program used Press and release the pwr prg power program button to select the program EP for E PAGE 96 Gels The digital display shows EP Mother E Base pwr prg Wy Eee button Ps n a time s y button digital display lt Bf ion LED To obtain the best results run the E PAGE 96 Gel immediately after removal from the pouch and loading Store and run E PAGE 96 Gels at room temperature Always load 10 20 ul deionized water first into all wells prior to sample loading For optimal results we do not recommend running reduced and non reduced samples on the same gel If you do choose to run these samples on the same gel avoid running reduced and non reduced samples in adjacent lanes as the reducing agent may have a carry over effect on the non reduced samples Avoid running samples containing different salt or protein concentrations in adjacent lanes Continued on next page 11 Loading E PAGE 96 Gels Continued Loading E PAGE Gels 12 Each E PAGE 96 Gel is supplied individually wrapped and ready for use Use short rigid tips for loading 1 2 3 Open the package and remove the E PAGE 96 Gel Remove the plastic comb from the gel Slide the gel into the two electrode connections on the Mother or Daughter E Base The two copper electrodes on the right side of the cassette must be in contact with the two electrode connections on
3. Continued Semi Dry 1 Ina clean container or Incubation Tray soak 4 pieces of Blotting 2 5 mm Blotting Filter Paper 8 6 cm x 13 5 cm in Protocol 2X NuPAGE Transfer Buffer page 21 Remove any air bubbles trapped between sheets using the Blotting Roller while the paper is still submerged in buffer 2 Ina clean container or Incubation Tray soak the E PAGE Blotting Pad in 2X NuPAGE Transfer Buffer page 21 Inspect the pad for air bubbles Press the pad to eliminate any visible air bubbles Note The Blotting Pad has no specific orientation either side may be facing the gel 3 Place 2 pieces of pre soaked 2 5 mm Blotting Filter Paper from Step 1 on the anode plate of a semi dry blotting apparatus Ensure that all filter paper sheets are aligned properly and remove any air bubbles with the Blotting Roller 4 Place the pre soaked blotting membrane see previous page on top of the filter paper stack and remove any air bubbles with the Blotting Roller 5 Remove the gel from the transfer buffer Gently rub a gloved finger over the well side of the gel to remove small gel pieces from the gel surface Re submerge the gel in transfer buffer to remove any gel pieces 6 Place the flat side of the gel on top of the blotting membrane well side up and remove any air bubbles with the Blotting Roller Fill the wells of the gel with 2X NuPAGE Transfer Buffer page 21 7 Place the pre soaked E PAGE Blotting Pad on the gel
4. 250 stain 0 015 in 30 ethanol and 10 acetic acid See note below e Destaining solution 8 acetic acid in deionized water e Clean microwave safe staining containers Do NOT use Incubation Tray Cat no LC2102 e 2 pieces of nylon membrane Cat no LC2003 e Rotary shaker e Microwave oven The volume of staining and destaining solutions needed will depend on the volume of the staining container The volume of all solutions must be sufficient to cover the gel completely When using the microwave staining protocol warm the staining and destaining solutions to 50 C without boiling It is important NOT to boil the solutions Since microwave ovens differ significantly we recommend testing various times at 10 second intervals and power settings of your microwave oven to achieve a temperature of 50 C in the volume of solution required for your particular staining container Perform these steps without the gel Once you have optimized the time and settings for your microwave oven use these settings for staining Continued on next page Coomassie R 250 Staining Protocol Continued Microwave For all staining and destaining steps described below be Staining sure to use sufficient reagents to completely cover the gel Protocol in a microwave safe container such that the gel moves freely during the staining and destaining steps 1 After electrophoresis remove the gel from the cassette page 17 and place the gel in
5. FREE at www invitrogen com epage Follow the instructions to download the software and user manual Methods Sample Preparation Note Materials Needed Prepare your protein samples as described in this section for electrophoresis on E PAGE 96 Gels We recommend that you read this section before preparing your samples Based on your method of detection choose a sample preparation method using the appropriate loading buffer Application Method Loading Buffer Routine staining and 1 page 7 Loading Buffer 1 western blotting Lumio Green 2 page8 Lumio Gel Detection Sample Buffer The E PAGE 96 Gels contain SDS and are designed for performing electrophoresis under denaturing conditions To obtain the best results we recommend performing SDS PAGE under reducing conditions If you need to perform SDS PAGE under non reducing conditions omit adding NuPAGE Sample Reducing Agent 10X during sample preparation e Protein sample e NuPAGE Sample Reducing Agent 10X e E PAGE Loading Buffer 1 4X included in the kit e Deionized water e Heating block set at 70 C e Molecular weight markers e Optional Lumio Green Detection Kit for detection of Lumio fusion proteins See page 33 for ordering information Continued on next page Sample Preparation Continued Amount of Protein Total Sample Volume High Salt or Detergent Samples Use up to 20 ug protein per well of the E PAGE 96
6. New Zealand 0800 600 200 Singapore 65 686 186 38 Taiwan 2 2651 6156 For other countries see our website www invitrogen com
7. a clean microwave safe container 2 Prepare Coomassie stain 0 015 Coomassie R 250 in 30 ethanol and 10 acetic acid See previous page 3 Add enough stain to completely cover the gel in the microwave safe container 4 Warm the gel and solution to about 50 C ina microwave oven see previous page Note Do NOT boil the solution 5 Incubate the gel in the warmed staining solution for 30 minutes on an orbital shaker at room temperature 6 Discard the stain rinse the gel briefly with water and discard the water 7 Add enough destaining solution see previous page to cover the gel during incubation 8 Place two pieces of positively charged nylon membrane on top of the destaining solution to speed up the destaining process 9 Warm the destaining solution gel and nylon membrane to 50 C in a microwave oven see previous page Note Do NOT boil the solution 10 Incubate the gel in the warm destaining solution on an orbital shaker at room temperature until the desired background is achieved Note To obtain a clear background perform destaining overnight 19 Drying E PAGE 96 Gels Drying the Gel 20 We recommend using the Large Gel Drying Kit available from Invitrogen to air dry the gel Refer to the Large Drying Kit manual or to the E PAGE Technical Guide at www invitrogen com or by contacting Technical Service page 34 Note The E PAGE 96 Gel will need at least 4 5 days for complete dryin
8. and gently roll out air bubbles with the Blotting Roller 8 Place 2 pieces of pre soaked 2 5 mm Blotting Filter Paper from Step 1 on top of the Blotting Pad Ensure that all filter paper sheets are aligned properly and flush with the gel membrane sandwich Remove any air bubbles trapped between sheets using the Blotting Roller 9 Place the cathode plate on the stack without disturbing the blot sandwich Follow the manufacturer s instructions to further assemble the semi dry blotting apparatus 10 Transfer at 25 V for 1h 19 V cm You may need to optimize the transfer conditions for your specific proteins or semi dry blot apparatus 23 Using the E Holder Platform Introduction The E Holder Platform is designed to hold E PAGE 96 Gels during loading Use the E Holder when you need to load multiple gels while the other gels are running on the E Base Note The E Holder is not a power supply unit cannot be connected to an electrical outlet and cannot be used to run E PAGE 96 Gels To obtain the best results run E PAGE 96 Gels on the Mother E Base or Daughter E Base within 15 minutes after loading the gel on E Holder Open the package and remove the gel Remove the comb from the E PAGE 96 cassette 3 Place the E PAGE 96 cassette in the E Holder Align the bottom left end of the cassette in the lower left alignment corner of the E Holder as shown below Proceed to loading the gel or to
9. cassette is correctly into base inserted and power is on a fan in the base will begin to run and a steady red light will be illuminated on the base Sample is overloaded Do not load more than 20 ug of protein sample per well For in gel staining load at least 200 ng protein per band Very low volumes of Load the recommended sample sample were loaded volume of 5 20 ul and always load 10 20 ul deionized water in all wells prior to sample loading Avoid introducing bubbles while loading the samples Bubbles will cause band distortion For proper band separation we recommend keeping sample volumes uniform and loading deionized water into empty wells Continued on next page Troubleshooting Continued Poor resolution Incorrect loading buffer Use the recommended loading or smearing of used buffers as described on page 3 bands 7 High salt or detergent Be sure the final concentration of concentration in samples salt or detergent in the sample is as described on page 5 You may need to manually increase the run time for high salt or detergent samples to obtain optimal results Gel was not For best results the gel should be electrophoresed run within 15 minutes of sample immediately after loading sample loading A1 tip not aligned Be sure to align the A1 tip properly prior to automated loading of E PAGE Gels page 10 Expired gel used Use properly stored gels before the specified expiration date
10. more details on setting the time or interrupting a run refer to the manual supplied with the E Base Note It is not necessary to have a gel in the Mother E Base if you are using a Daughter E Base However the Mother E Base must be plugged into an electrical outlet 1 To begin electrophoresis press and release the pwr prg power program button located on the lower right corner of the Mother E Base If you are using a Daughter E Base press and release the pwr prg button located on the lower right corner of the Daughter E Base x Mother E Base faen x E v Daughter E Base D x a a The red light will change to a green light and the digital display will show the count down time during the run Continued on next page 13 Electrophoresis of E PAGE 96 Gels Continued i While the run is in progress you can add to the run Using an progress y E B ase time by pressing the time button to select the desired continue d time and then release the time button The recommended running time for an E PAGE 96 Gel is 14 minutes Avoid running an E PAGE 96 Gel for more than 25 minutes To interrupt or stop a run in progress see next page Note If your sample contains high salt or detergent concentrations you may need to manually increase the run time For electrophoresis of Lumio fusion proteins we recommend running the gel for an additional 2 minutes to prevent the formati
11. optimal capture of small proteins Appendix Accessory Products Additional Ordering information for additional products available Products separately from Invitrogen is provided below For details contact Technical Service next page or visit www invitrogen com Product Quantity Catalog no Mother E Base 1 EB M03 Daughter E Base 1 EB D03 E PAGE 48 Gels 8 pk EP048 08 E PAGE 48 Starter Kit 1 kit EPST48 08 E Holder Platform 2 EH 03 E PAGE Loading Buffer 1 4X 4 5 ml EPBUF 01 E PAGE Blotting Pad 4 pk LC2101 NuPAGE Sample Reducing Agent 10X 10 ml NP0009 E PAGE SeeBlue Pre stained Protein Standard 500 pl LC5700 E PAGE MagicMark Unstained Protein Standard 250 pl LC5701 BenchMark Fluorescent Protein Standard 125 ul LC5928 NuPAGE Transfer Buffer 20X 125 ml NP0006 NuPAGE Antioxidant 15 ml NP0005 Blotting Roller 1 LC2100 Nitrocellulose Filter Paper Sandwich 0 45 um 16 pk LC2006 Nitrocellulose Filter Paper Sandwich 0 2 um 16 pk LC2009 Invitrolon PVDF Filter Paper Sandwich 0 45 um 16 pk LC2007 Blotting Filter Paper 2 5 mm 50 pk LC2008 Incubation Trays 8 pk LC2102 Lumio Green Detection Kit 1 kit LC6090 Large Gel Drying Kit 1 kit NI2207 Gel Dry Drying Solution 1X 500 ml LC4025 WesternBreeze Chromogenic Kit Anti Mouse 1 kit WB7103 WesternBreeze Chemiluminescent Kit Anti Mouse 1 kit WB7104 33 Technical Service World Wide Visit the Invitrogen Web Res
12. sonendeceesen decvehesteseseleserersanglsassansecvenerse 25 Using E Editor 2 02 Software ijasistccceiscatendsasiestassateitealainna tavadsities 29 Troubleshooting nasterio nieis AE EE S EE E Eii 30 aN aeo ID A E E A E T N E A 33 Accessory Productes i etic de att shh atte aa E AEAT a 33 Technical Servissi eea siae ie EE EERE ES Aah eee sees 34 Purchaser Notification asresten E mam E 36 iii iv E PAGE Experienced Users Procedure Introduction This quick reference sheet is included for experienced users of E PAGE 96 Gels If you are a first time user follow the detailed protocols provided in this manual Note For optimal results load each E PAGE 96 Gel within 30 minutes of removing the gel from the plastic pouch and run within 15 minutes of loading Prepare Use up to 20 ug protein per lane of the E PAGE 96 Gel See Sample page 4 for sample preparation Align Robotic If you are using automated loading align the robotic tip Tip Assembly assembly as described below 1 Set position of the first tip approximately 1 mm above the slope of the A1 well Figure 2 page 10 to ensure that the remaining tips are aligned above the slopes of the other wells Refer to the manufacturer s manual for your automated liquid handling system to program this setting Proceed to loading the gel Select Program Plug the Mother E Base into an electrical outlet and Load 2 If using a Daughter E Base connect the Daughter E Base t
13. staining or blotting we recommend using the E PAGE Loading Buffer 1 4X included in the kit for preparing samples The E PAGE Loading Buffer 1 is optimized for E PAGE 96 Gels Do not use any other SDS PAGE sample buffer e SDS PAGE and In Gel Detection of Lumio Fusion Proteins Method 2 page 8 For detection of Lumio fusion proteins with the Lumio Green Detection Kit use the Lumio Gel Sample Buffer 4X supplied with the Lumio Green Detection Kit This buffer is specifically formulated to provide optimal results with the Lumio Green Detection Reagent Do not use E PAGE Loading Buffer 1 to prepare samples for Lumio Green Detection Continued on next page Sample Preparation Continued Molecular Weight Standards Method 1 Routine Staining and Blotting To obtain the best results we recommend using the following protein molecular weight standards See page 25 for molecular weight standard calibration in E PAGE 96 Gels See page 33 for ordering information Marker E PAGE SeeBlue Pre stained Protein Standard E PAGE MagicMark Unstained Protein Standard TM BenchMark Fluorescent Protein Standard Amount Application 10 ul Electrophoresis 5 pl Western Blotting 5 ul Lumio Detection Use this protocol if you are performing SDS PAGE followed by routine staining or blotting If the E PAGE Loading Buffer 1 4X is stored at 4 C bring the buffer to ro
14. the A1 well Figure 2 below This will ensure that the remaining tips are aligned above the slopes of the remaining wells Refer to the manufacturer s manual of your automated liquid handling system to program this setting After programming the setting load your samples During loading the samples will fall onto the slopes of the wells and be drawn into the wells by capillary force Figure 1 4 5 6 7 8 9 10 11 12 M aa aa aa aa oo OOP DOH ODO oo a aa aada aa aa aa ag O 3 3 3 3 ODO ODO O ODCODOD a a aa a a aa a aa aa aada aag OOO OO OO OO OD OO CO a a aa a a aa aa aa a aada oo Ir nm Oo O U gt OOD ODP CODO ODO OCO ff Figure 2 Fd Continued on next page 10 Loading E PAGE 96 Gels Continued Selecting a Program on E Base Important The recommended program for electrophoresis of the E PAGE 96 Gel is program EP which runs for 14 minutes Brief instructions for using the E Base are included in the following sections For further details refer to the E Base manual available at www invitrogen com You need to select program EP on the base prior to inserting a gel 1 ae h to electrical outlet gt Plug the Mother E Base into an electrical outlet using the electrical plug on the base If using Daughter E Base connect the Daughter E Base to a Mother E Base or another Daughter E Base connected to a Mother E Base
15. the base E PAGE 96 Gel k Mother E Base When the gel is properly inserted into the base a fan in the base begins to run a red light illuminates at the lower left corner of the base and the digital display shows 14 minutes for program EP or last time setting Note If you accidentally inserted a cassette into the base before selecting program EP remove the cassette select program EP and reinsert the cassette To add to the run time of 14 minutes press the time button until the desired time is displayed and then release the time button Load deionized water to each well of the E PAGE 96 Gel prior to loading your samples or protein molecular weight standards Load samples into the gels using a multichannel pipetter or a liquid handling system as described below First Load Deionized Water Then Load Sample in Loading Buffer 20 ul 5 10 ul 19 10 ul 11 20 ul Load the appropriate protein molecular weight marker in the marker wells of the gel See page 7 for recommended molecular weight standards Proceed immediately to Electrophoresis next page Electrophoresis of E PAGE 96 Gels Introduction Using an E Base After loading your protein samples on the E PAGE 96 Gels proceed immediately to electrophoresis using the E Base TM See page 26 for electrophoresis results of E PAGE 96 Gels Instructions for running an E PAGE 96 Gel in a Mother E Base or Daughter E Base are provided below For
16. Gel The amount of protein required will depend on the staining or western detection method used for visualizing proteins after electrophoresis If you are unsure of how much to use test a range of concentrations to determine the optimal concentration for your sample The maximum recommended loading amount for E PAGE 96 Gel is 20 ug protein per well Excess proteins will cause poor resolution Note To ensure a proper LDS lithium dodecyl sulfate from Loading Buffer 1 to protein ratio limit sample protein or lipid from the sample amount to 2 ug ul of the final sample volume T The recommended total sample volume for E PAGE 96 Gels is 10 ul If desired you may load between 5 20 ul of sample For best results avoid loading less than 5 ul of sample and more than 20 ul of sample Prior to sample loading load 10 20 ul deionized water first into all wells Samples containing high salt or detergents will cause loss of resolution on E PAGE 96 Gels Dilute the samples such that the final concentration of the salt or detergent in the sample is as described below Salt Detergent Final Concentration Triton X 100 lt 0 5 Tween 20 lt 0 5 SDS lt 4 Tris lt 200 mM NaCl lt 250 mM Continued on next page Sample Preparation Continued Loading Buffer Use the appropriate loading buffer based on the application as described below e SDS PAGE and Routine Staining Method 1 page 7 For SDS PAGE and
17. Sample leaking Sample is overloaded or Be sure to load the recommended from the wells wells are damaged volume of sample per well page 5 Remove the comb carefully without damaging the wells Over run the gel Accidentally selected an Select program EP for or need more incorrect program E PAGE 96 Gels time to run gel If you accidentally selected an incorrect program and are at the beginning of the run stop the run and select the desired program If you are well into the run check the gel to see where the loading dye is running Estimate the amount of time remaining and then manually stop the run Continued on next page 31 Troubleshooting Continued Be sure that the E PAGE Blotting Problem Cause Protein bands Non uniform electric distorted on membrane after semi dry blotting Weak transfer of high molecular weight samples during semi dry blotting Weak transfer of low molecular weight samples 32 field created around wells Incorrect gel orientation Blot stack disturbed during or after assembly Not enough SDS in sample Use of large pore membranes allow small proteins to blow through Pad is used correctly Be sure that the well side of the gel is not facing the membrane Take care not to disturb the blot stack Make sure transfer buffer contains no methanol for transferring TM E PAGE 96 Gels Use 0 2 um nitrocellulose membrane for
18. Step 4 for automated liquid handling system loading Procedure Prp Alignment corner Note The E PAGE 96 Gel will not fit into the E Gel 96 Holder previously available from Invitrogen due to the tabs on the E PAGE 96 cassette 4 Optional Set up your automated liquid handling system to load samples into the gel placed on an E Holder Program your system to load the samples approximately 5 minutes before the previous electrophoresis run is complete This will ensure that the loaded gel on the E Holder will be placed onto a Mother E Base or Daughter E Base within the recommended time of 15 minutes 24 Expected Results Molecular The apparent molecular weight values shown below are Weight derived from the construction of a calibration curve in the Calibration E PAGE 96 buffer system Use the values listed in the figures below for the most accurate calibration of your protein on an E PAGE 96 Gel E PAGE SeeBlue Chemiluminescent image of Pre stained Protein E PAGE MagicMark Standard Molecular Weight Western Protein Standard on E PAGE 96 6 Gel ei 1 261kDa wee 11 220kDa _ 2 2 173kDa 2 120kDa A 3 97 kDa p i p ui _ 4 42kDa a 3 a 5 21kDa 5 20kDa co 4 4 we 5 a Continued on next page 25 Expected Results Continued TM Electrophoresis Electrophoresis results obtained using an E PAGE 96 6 Results Gel are shown below E PAGE SeeBlue Pre stained Protein Sta
19. age Continued on next page 34 Technical Service Continued Limited Warranty Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service please contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to the specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no
20. al Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Continued on next page Purchaser Notification Continued Limited Use Label License No 71 NuPAGE Bis Tris Gel System This product is the subject of one or more of US P
21. atent Nos 5 578 180 5 922 185 6 059 948 6 096 182 6 143 154 6 162 338 and EP0753142 owned by Invitrogen The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are r
22. e compatible with a multichannel pipetter or 8 12 or 96 tip automated liquid handling devices E PAGE 96 pre cast Gels are qualified by running E PAGE SeeBlue Pre Stained Protein Standards and BSA under standard running conditions as described in this manual Gels are visualized for proper resolution and migration of bands Visual inspection is also performed to ensure that the gels are free from bubbles spots and any gel residues The E PAGE SeeBlue Pre Stained Standard must show 5 distinct bands when separated by electrophoresis on an E PAGE 96 6 Gel Overview Introduction Applications E PAGE 96 Pre cast Gels Introduction The E PAGE High Throughput HTP Protein Electrophoresis System is designed for fast high throughput protein electrophoresis in a horizontal format The E PAGE 96 System consists of the following components e E PAGE 96 Pre cast Gels e E Base Electrophoresis Device e E PAGE Loading Buffer e E Holder Platform e E Editor 2 02 Software The E PAGE HTP Protein Electrophoresis System is ideal for screening protein samples using these applications e In gel staining with Lumio Green Reagent e Staining Coomassie silver or fluorescent stains e Western blotting e Functional assays E PAGE 96 Gels are self contained pre cast gels that include a buffered gel matrix and electrodes packaged inside a disposable UV transparent ca
23. en is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 2003 2005 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use Coomassie is a registered trademark of Imperial Chemical Industries PLC Windows is a registered trademarks of Microsoft Corporation Triton X 100 is a registered trademark of Rohm amp Haas Co Tween 20 is a registered trademark of Atlas Chemical Co Alphalmager is a trademark of Alpha Innotech Corporation e 3 Invitrogen life technologies Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Tel 1 760 603 7200 Tel Toll Free 1 800 955 6288 Fax 1 760 602 6500 Email tech_service invitrogen com European Headquarters Invitrogen Ltd Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF UK Tel Free Phone Orders 0800 269 210 Tel General Enquiries 44 0 141 814 6100 Fax 44 0 141 814 6260 Email eurotech invitrogen com International Offices Argentina 5411 4556 0844 Australia 1 800 331 627 Brazil 55 11 5051 7422 Canada 800 263 6236 China 10 6849 2578 Hong Kong 2407 8450 Japan 03 3663 7974
24. esold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Ave Carlsbad CA 92008 Phone 760 603 7200 Fax 760 602 6500 37 Purchaser Notification Continued Limited Use Label License No 264 E PAGE Gels 38 This product is the subject of one or more of US Patents and foreign equivalents licensed by Invitrogen Corporation The purchase or transfer of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use th
25. g Due to the thickness of the E PAGE 96 Gel vacuum drying is not recommended and may cause the gel to crack Semi Dry Blotting of E PAGE 96 Gels Introduction Materials Needed 2X NuPAGE Transfer Buffer A semi dry blotting procedure for blotting E PAGE Gels is described in this section You will need a semi dry transfer apparatus that is capable of accommodating the dimensions of an E PAGE Gel 8 6 cm x 13 5 cm and a power supply For details on semi wet blotting refer to the E PAGE Technical Guide available at www invitrogen com or by contacting Technical Service page 34 See page 27 for results obtained with semi dry blotting of E PAGE 96 Gels You will need the following items See page 33 for ordering information e Semi dry blotter e NuPAGE Transfer Buffer 20X e NuPAGE Antioxidant e Blotting membranes Invitrolon Filter Paper Sandwiches or Nitrocellulose Membrane Filter Paper Sandwiches e E PAGE Blotting Pad supplied with E PAGE Gels or available separately e 4 pieces of 2 5 mm Blotting Filter Paper e Blotting Roller e Incubation Tray We recommend using 2X NuPAGE Transfer Buffer with NuPAGE Antioxidant without methanol for the optimal transfer of most proteins from E PAGE 96 Gels For one gel prepare 500 ml of 2X NuPAGE Transfer Buffer as follows Buffer Component 2X NuPAGE Transfer Buffer NuPAGE Transfer Buffer 20X 50 ml NuPAGE A
26. hMark Fluorescent Protein Standard 5 ul 2 E coli CAT Lumio fusion protein 10 ul 3 E coli kinase Lumio fusion protein 10 ul Figure 2 Lane Sample 1 BenchMark Fluorescent Protein Standard 5 ul 2 48 kDa Lumio fusion protein 25 pmole 3 48 kDa Lumio fusion protein 5 pmole 4 48 kDa Lumio fusion protein 1 pmole Using E Editor 2 02 Software Introduction E Editor 2 02 software for Windows allows you to reconfigure digital images of E PAGE 96 Gels for analysis and documentation E Editor 2 02 reconfigures the wells of an E PAGE 96 Gel into a side by side format for easy comparison and analysis You can reconfigure gels that were scanned in the original gel cassette or gels that were removed from the cassette and stained or blotted You can also group the images of multiple gels loaded from a 384 well microtiter plate into a single image with a layout corresponding to that of the original plate Capture an image of the gel as described below and use the E Editor 2 02 software to e Align and arrange the lanes in the image e Save the reconfigured image for further analysis e Copy and paste selected lanes or the entire reconfigured image into other applications for printing saving emailing and or publishing Guidelines Guidelines are provided below to image your E PAGE 96 Gel for Imaging after staining for analysis with E Editor 2 02 software e Usea flatbed scanner or d
27. igital camera to capture a digital TM image of your E PAGE 96 gel e Align the gel properly while imaging i e not at an angle and make sure that the gel features are clear and distinct in the image e Image the gel using a resolution of 150 dpi or higher e Scan the gel cassette well side up if the gel is inside the cassette e Scan the gel well side up and fill the wells with water if the gel is removed from the cassette to minimize interference from the wells in the image e Use a flatbed scanner with top illumination for best results 29 Troubleshooting Trouble shooting Problem No current Poor resolution or smearing of bands 30 The table below provides some solutions to possible problems you might encounter during the electrophoresis of E PAGE 96 Gels For troubleshooting problems with E Base and staining refer to the manuals supplied with the appropriate products Daughter E Base used Do not use the Daughter E Base without a Mother without a Mother E Base The E Base Daughter E Base does not have an electrical plug to connect to an electrical outlet Copper contacts inthe Make sure that the copper base are damaged due contacts in the base are intact to improper use Expired or defective gel Use properly stored gels before cassette used the specified expiration date E PAGE 96 cassette is Remove cassette and reinsert not correctly inserted When the
28. is product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrog
29. ls E PAGE Loading Buffer 1 4X 4 5 ml Butterfly Opener 1 E PAGE Blotting Pad 1 E PAGE 96 Starter Kit Quantity E PAGE 96 6 Gels 4 gels E PAGE Loading Buffer 1 4X 4 5 ml Butterfly Opener 1 E PAGE Blotting Pad 1 Mother E Base 1 E PAGE SeeBlue Pre stained Protein 500 ul Standard Shipping and The E PAGE 96 Gels Loading Buffer 1 4X E PAGE Storage Blotting Pad and Mother E Base are shipped at room temperature E PAGE SeeBlue Pre stained Protein Standard is shipped on blue ice Upon receipt store the contents as described below e Store E PAGE 96 Gels E PAGE Blotting Pad and Mother E Base at room temperature Do not allow the temperature to drop below 4 C or rise above 40 C when storing the gels e Store E PAGE Loading Buffer 1 4X at room temperature or at 4 C e Store E PAGE SeeBlue Pre stained Protein Standard at 4 C vil Specifications Specifications Product Qualification viii Each E PAGE 96 Gel contains 96 sample wells and 8 marker wells M Cassette Size 13 5 cm 1 x 10 8 cm w x 0 67 cm thick Gel Formulation Proprietary operating at neutral pH Separation Range 10 300 kDa Gel Thickness 3 7 mm Gel Volume 50 ml Well Depth 3 mm Well Opening 3 8 mm x 1 8 mm Well Bottom 3 3 mm x 1 1 mm Running Distance 16 mm one well to the next Space between Wells 9 mm The well openings of the E PAGE 96 cassette ar
30. m page 24 If you are using an automated liquid handling device it is important to align the robotic tip loading assembly to the proper setting prior to loading samples on the E PAGE 96 Gel This ensures proper loading of samples into the wells See next page for more details e Dispose of gels as hazardous waste e Avoid touching the gel while the gel is running Each E PAGE 96 Gel is labeled with an individual barcode The barcode facilitates the identification of each gel cassette during electrophoresis of multiple gels Each E PAGE 96 Gel contains an EAN 39 type of barcode which is recognized by the majority of commercially available barcode readers Refer to the manufacturer s instructions to set up the barcode reader Note When capturing an image of the E PAGE 96 Gel note that the barcode label is easily overexposed To ensure that the barcode label is distinct and readable in the image experiment with different shutter settings for your particular camera Continued on next page Loading E PAGE Gels Continued Aligning the The wells of the E PAGE 96 Gel are staggered to provide Robotic maximum run length Figure 1 below For proper sample Loadin loading it is important to program your automated liquid A handling system to set the A1 tip of the 8 12 or 96 tip ssembly robotic head over the E PAGE 96 Gel as described below Set the position of the first tip approximately 1 mm above the slope of
31. ndard 10 pl was loaded into alternate sample wells and 8 marker wells The gel was electrophoresed for 14 minutes using standard conditions described in this manual Note The wells of an E PAGE 96 Gel are staggered Protein bands migrate between adjacent wells in the row below For example the bands of lane A3 will migrate between wells B2 and B3 Continued on next page 26 Expected Results Continued Western Western blotting results obtained using an E PAGE 96 6 Blotting Gel are shown below Results E PAGE MagicMark Unstained Protein Standard 5 ul was loaded into alternating sample wells The gel was electrophoresed for 14 minutes using standard conditions described in this manual Proteins were transferred to a 0 45 um nitrocellulose membrane by semi dry blotting as described Detection was performed with WesternBreeze Anti Mouse Immunodetection Kit using 1 5000 dilution of Anti V5 primary antibody from Invitrogen Continued on next page 27 Expected Results Continued Results with Lumio Green Reagent 28 Results with Lumio detection on an E PAGE 96 6 Gel using the Lumio Green Detection Kit are shown below The gel in Figure 1 was visualized and imaged on a UV transilluminator Alphalmager Imaging System with an ethidium bromide filter using a 2 second exposure and the gel in Figure 2 was visualized using a 4 second exposure Figure 1 Lane Sample 1 Benc
32. ntioxidant 0 5 ml Deionized Water to 500 ml Continued on next page 21 Semi Dry Blotting of E PAGE 96 Gels Continued Equilibrating the Gel Preparing Blotting Membrane 22 Equilibration of the gel in 2X Transfer Buffer results in removal of salts that may increase conductivity and heat during transfer Perform equilibration for the recommended time as longer equilibration will result in protein diffusion 1 After electrophoresis remove the gel from the cassette as described on page 17 2 Using the Butterfly Opener or a gel knife trim off the top and bottom electrode areas of the gel 3 Equilibrate the E PAGE Gel in 200 ml 2X NuPAGE Transfer Buffer see previous page for 30 minutes Perform this equilibration on a shaker Nitrocellulose 1 Use pre cut Nitrocellulose Filter Paper Sandwich or cut nitrocellulose membrane to the appropriate size 8 6 cm x 13 5 cm 2 Soak the membrane in 2X NuPAGE Transfer Buffer see previous page for several minutes in the Incubation Tray PVDF 1 Use pre cut Invitrolon Filter Paper Sandwich or cut PVDF membrane to the appropriate size 8 6 cm x 13 5 cm 2 Pre wet the membrane for 30 seconds in methanol ethanol or isopropanol Briefly rinse in deionized water 3 Soak the membrane in 2X NuPAGE Transfer Buffer see previous page for several minutes in the Incubation Tray Continued on next page Semi Dry Blotting of E PAGE 96 Gels
33. o a Mother E Base or another Daughter E Base connected to a Mother E Base Press and release pwr prg power program button to select the program EP for E PAGE 96 Gels Remove gel from the package and remove the plastic comb from the gel Slide the gel into the two electrode connections on the Mother E Base or Daughter E Base Load samples into the gels using a multichannel pipetter or an automated liquid handling system First load deionized water into each well and then load your samples as described below First Load Deionized Water Then Load Sample in Loading Buffer or MW marker 20 pl 5 10 pl 19 10 ul 11 20 ul Load the appropriate protein molecular weight marker in the marker wells of the gel Continued on next page v E PAGE Experienced Users Procedure Continued Step Action Electrophoresis 1 Press and release the pwr prg button located on the with E Base lower right corner of the base to begin electrophoresis 2 The Mother E Base and Daughter E Base will signal the end of the run with a flashing red light and rapid beeping for 2 minutes followed by a single beep every minute 3 Press and release the pwr prg button to stop the beeping 4 Remove the E PAGE 96 cassette from the base 5 Open the gel cassette for staining or blotting applications Opening the Open the E PAGE 96 cassette with the Butterfly Opener Cassette included in the kit and remove the gel 1 Inse
34. om temperature and mix briefly prior to use 1 Prepare your samples in a total volume of 10 pl in E PAGE Loading Buffer 1 as described below If you need to prepare samples in a volume of 5 20 ul adjust the volume accordingly Reagent Reduced Non Reduced Protein Sample x ul x pl E PAGE Loading 2 5 ul 2 5 ul Buffer 1 4X NuPAGE Sample 1 ul Reducing Agent 10X Deionized Water to 10 ul to 10 ul 2 Incubate the samples at 70 C for 10 minutes 3 Proceed to Loading E PAGE 96 Gels page 9 Continued on next page Sample Preparation Continued Method 2 Lumio Detection Use this protocol to prepare samples for specific detection of Lumio fusion proteins with the Lumio Green Detection For details on Lumio Green Detection Kit refer to the product manual at www invitrogen com or contact Technical Service page 34 Refer to the Lumio Detection manual for details on each type of protein Prepare protein samples as follows Protein Sample Sample Lumio Gel Sample Volume Buffer 4X Volume Bacterial samples 15 pl 5 ul Mammalian lysate 15 ul 5 ul Partially purified 15 ul 5 pl sample Purified sample 15 pl 5 ul In vitro expressed 20 ul Not needed There is no need to add Lumio Gel Sample Buffer 4X as the sample is already prepared in this buffer Thaw the Lumio Green Detection Reagent and mix well To the protein sample
35. on of a fluorescent dye front 2 The Mother E Base and Daughter E Base signals the end of the run with a flashing red light and rapid beeping for 2 minutes followed by a single beep every minute At the end of the run the digital display shows the original time setting not any time change that was made during the electrophoresis The digital display also shows the elapsed time up to 19 minutes with a negative sign since the end of the run 3 Press and release the pwr prg button to stop the beeping The light turns to a steady red and the digital display shows the last time setting 4 Remove the gel cassette from the Mother E Base and Daughter E Base Note The bands in the gel will diffuse within 40 minutes 5 For staining or blotting the gel see pages 18 23 For visualizing Lumio fusion proteins see page 16 Continued on next page 14 Electrophoresis of E PAGE 96 Gels Continued Interrupting a Run Note Maintaining E Base You can interrupt an electrophoresis run at any time by pressing and releasing the pwr prg button to stop the current The stopped current is indicated by a steady red light and the digital display will flash to indicate that the run has been interrupted You can remove the gel from the base to check the progress of the run Then e To continue the run from the point at which it was stopped reinsert the gel and press and release the pwr prg button The ligh
36. ource using your World Wide Web Web browser At the site you can e Get the scoop on our hot new products and special product offers e View and download vector maps and sequences e Download manuals in Adobe Acrobat PDF format e Explore our catalog with full color graphics e Obtain citations for Invitrogen products e Request catalog and product literature Once connected to the Internet launch your Web browser Internet Explorer 5 0 or newer or Netscape 4 0 or newer then enter the following location or URL http www invitrogen com and the program will connect directly Click on underlined text or outlined graphics to explore Don t forget to put a bookmark at our site for easy reference Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters European Headquarters Invitrogen Corporation Invitrogen Ltd 1600 Faraday Avenue Inchinnan Business Park Carlsbad CA 92008 USA 3 Fountain Drive Tel 1 760 603 7200 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Tech Fax 44 0 141 814 6117 E mail tech_service invitrogen com E mail eurotech invitrogen com MSDS To request an MSDS visit our Web site at Information www invitrogen com On the home page go to Technical Resources select MSDS and follow instructions on the p
37. refer to the Lumio Green detection kit manual available at www invitrogen com or by contacting Technical Services page 34 After electrophoresis is complete immediately visualize and image the gel as described below There is no need to remove the E PAGE 96 Gel from the cassette to visualize Lumio fusion proteins 1 Place the cassette on a UV transilluminator equipped with a camera and select the ethidium bromide or SYBR Green filter on the camera You may also use a laser based scanner with a laser line that falls within the excitation maxima of the stain 500 nm and a 535 nm long pass filter or a band pass filter centered near the emission maxima of 535 nm Note Adjust the settings on the camera prior to turning on the UV transilluminator Avoid exposing the gel to UV light for long periods of time 2 Image the gel with a suitable camera with the appropriate filters using a 4 10 second exposure You may need to adjust the brightness and contrast to reduce any faint non specific bands You should see fluorescent bands of Lumio fusion proteins and the gel should have minimum background as shown on page 28 Opening the E PAGE 96 Cassette Introduction Opening the Cassette Note Prior to staining or blotting the E PAGE 96 Gel you need to open the cassette using the red plastic Butterfly Opener and remove the gel Insert the wide side of the red Butterfly Opener included in the kit between the
38. responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness fora particular purpose 35 Purchaser Notification Limited Use Label License No 61 E Gel Agarose Gels 36 This product is the subject of one or more of U S Patent Nos 5 582 702 5 865 974 6 379 516 and 6 562 213 and foreign equivalents owned by Invitrogen Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commerci
39. rt the wide side of the red Butterfly Opener between the tabs at the edge of the E PAGE 96 cassette and twist to separate the two halves of the cassette 2 Pull apart the cassette halves with your hands until the cassette halves are separated 3 Using the Butterfly Opener or a gel knife trim the top and bottom electrode areas of the gel 4 Proceed to staining or blotting Staining and e For Coomassie R 250 staining see page 18 Blotting e For semi dry blotting see page 21 E PAGE 96 gels may be stained using many protein staining methods For details on other staining and blotting methods refer to the E PAGE Technical Guide at www invitrogen com or by contacting Technical Service page 34 Using 1 Use an appropriate digital documentation system to E Editor 2 02 capture a digital image of the gel Software 2 Download E Editor 2 02 software and the instruction manual for free at www invitrogen com epage 3 Use the E Editor 2 02 software to align and arrange the lanes in the image and save the reconfigured image for further analysis vi Contents and Storage Types of This manual is supplied with the following products Products Product Quantit Catalog no y g E PAGE 96 6 Gels 8 pack EP096 06 E PAGE Starter Kit 1 kit EPST96 06 Contents The contents for E PAGE 96 Gels and E PAGE Starter Kit are listed below E PAGE 96 Gels Quantity E PAGE 96 6 Gels 8 ge
40. s from Step 1 add 0 2 ul Lumio Green Detection Reagent in a fume hood Mix well Return the Lumio Green Detection Reagent to 20 C immediately after use Incubate samples at 70 C for 10 minutes Allow samples to cool for 1 2 minutes and centrifuge briefly at maximum speed in a microcentrifuge Thaw the Lumio In Gel Detection Enhancer and mix well Add 2 ul Lumio In Gel Detection Enhancer to the samples Mix well and incubate the samples at room temperature for 5 minutes Return the Lumio In Gel Detection Enhancer to 20 C immediately after use Proceed to Loading E PAGE 96 Gels page 9 Note For electrophoresis of Lumio fusion proteins we recommend running the gel for an additional 2 minutes to prevent the formation of a fluorescent dye front After electrophoresis is complete immediately visualize and image the gel as described on page 16 Loading E PAGE 96 Gels Introduction Using the Barcode After preparing your samples you are ready to load the E PAGE 96 Gel This section describes the procedure for loading protein samples and molecular weight standards on E PAGE 96 Gels The Mother E Base and Daughter E Base are designed to fit most robotic platforms allowing you to load and run E PAGE 96 Gels directly on an automated liquid handling system If you need to load multiple gels on a robotic platform while other gels are running on the E Base use an E Holder Platfor
41. ssette Each E PAGE 96 Gel contains 96 sample lanes and 8 marker lanes in a patented staggered well format that is compatible for loading with a multichannel pipetter or with an automated liquid handling system in the standard 96 well plate format see page 10 for specifications After electrophoresis the E PAGE cassette is easily opened with the included Butterfly Opener to remove the gel for staining or blotting applications In addition each E PAGE 96 cassette is labeled with an individual barcode to facilitate identification of the gel using commercial barcode readers page 9 Continued on next page Overview Continued Diagram of an A diagram of the E PAGE 96 gel cassette is shown below E PAGE 96 0 electrode Cassette 4 AA ___ staggered well format tabs for opening gel cassette barcode copper i a electrode E PAGE 96 ss sssssr 0302 20 Invitrogerr E Base E PAGE 96 Gels are used with specially designed electrophoresis device that combines a base and a power supply Two types of devices are available from Invitrogen e The Mother E Base catalog no EB M03 has an electrical plug that can be connected directly to an electrical outlet and is used for electrophoresis of one E PAGE 96 Gel The Mother E Base has been tested for electrophoresis with up to three Daughter E Bases connected at one time TM e The Daughter E Base catalog no EB D03 connec
42. t changes to steady green and the digital display shows the count down time e To cancel the rest of the interrupted run press and hold the pwr prg button for a few seconds The digital display will reset and the base will return to Ready Mode If desired you can then program a new run time and rerun the gel In case of an external power failure the run will continue when the power resumes The Mother E Base and Daughter E Base will signal the end of the run as described on the previous page except the light will be an alternating red green to indicate that an external power failure has occurred during the run We recommend that you disconnect the Mother E Base from the outlet when not in use for a prolonged period of time Keep the surfaces of the Mother E Base and Daughter E Base free of contaminants To clean disconnect bases from power source and wipe with a dry cloth Do not attempt to open or service the bases To honor the warranty bases should only be opened and serviced by Invitrogen 15 Visualizing Lumio Fusion Proteins Introduction Visualizing Lumio Fusion Proteins 16 The steps involved in detecting Lumio fusion proteins run on an E PAGE 96 Gel are described below To visualize Lumio fusion protein bands in the gel after electrophoresis you will need a UV transilluminator or a laser based scanner see below For further details on imaging Lumio fusion proteins
43. tabs at the edge of the E PAGE 96 cassette and twist to separate the two halves of the cassette Figure A below Gently pull apart the cassette halves with your hands until the cassette halves are completely separated and the gel is exposed Figure B below Carefully remove the gel from the cassette Using the Butterfly Opener or a gel knife trim the top and bottom electrode areas of the gel Proceed to staining next page or blotting page 21 Small pieces of gel material may remain in the wells of an E PAGE Gel after removal of the gel from the cassette To obtain the best staining and blotting results remove any small pieces of gel material in the wells of the gel by gently rubbing a gloved hand over the well side of the gel 17 Coomassie R 250 Staining Protocol Introduction Additional Staining Protocols Materials Needed Note Important 18 The instructions for visualizing protein bands on E PAGE 96 Gels with Coomassie R 250 stain using a microwave protocol are described in this section Using the microwave protocol reduces the amount of time needed for staining and destaining You may stain E PAGE 96 Gels using many protein staining methods For additional staining protocols refer to the E PAGE Technical Guide available at www invitrogen com or by contacting Technical Service page 34 You will need the following items for staining one E PAGE 96 Gel e Coomassie R
44. ts to the Mother E Base and together they can be used for the independent electrophoresis of 2 or more E PAGE 96 Gels The Daughter E Base does not have an electrical plug and cannot be used without a Mother E Base Refer to page 13 for a diagram of the bases Important The E PAGE 96 Gel is not compatible with the E Gel 96 bases cat nos G7100 01 G7200 01 previously available from Invitrogen The older E Gel 96 bases do not have the E Base inscription on the platform Continued on next page Overview Continued Loading Buffer E Holder Platform E Editor 2 02 Software E PAGE 96 Gels are supplied with E PAGE Loading Buffer 1 4X which is optimized for the E PAGE System E PAGE Loading Buffer 1 4X is recommended for routine SDS PAGE and staining or blotting applications Do not use any other sample buffer The E Holder Platform is designed to hold E PAGE gels during loading Use the E Holder when you need to load multiple gels while other gels are running on the E Base Note The E Holder is not a power supply unit cannot be connected to an electrical outlet and cannot be used to run gels See page 24 for more information about the E Holder Platform The E Editor 2 02 Software allows you to quickly reconfigure digital images of E PAGE 96 results for analysis and documentation page 29 E Editor 2 02 software can be downloaded
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