E-PAGE™ 48 Protein Electrophoresis System
Contents
1. E Editor 2 02 Software iBlot Gel Transfer Device 30 The following E PAGE Gel kits are available from Life Technologies Product Quantity Catalog no E PAGE 48 8 Gels 8 pack EP048 08 E PAGE 96 6 Gels 8 pack EP096 06 The following electrophoresis bases are available from Life Technologies for running E PAGE gels e The Mother E Base Device Cat no EB M03 is used for electrophoresis of one E PAGE Gel e The Daughter E Base Device Cat no EB D03 attaches to the Mother E Base Device and together are used for the independent electrophoresis of two or more E PAGE Gels The E Holder Platform is used to hold an E PAGE Gel in place while loading Ordering information can be found on the following page The E Editor 2 02 software is available FREE with the purchase of any E PAGE Gels or related equipment The software may be downloaded from www lifetechnologies com epage The iBlot Gel Transfer Device is available from Life Technologies for transfer of proteins from E PAGE gels to nitrocellulose or PVDF membranes Product Catalog no iBlot Gel Transfer Device 1 unit IB1001 IB1001UK IB1001EU iBlot Gel Transfer Stack Nitrocellulose Regular 10 pack 1B3010 01 iBlot Gel Transfer Stack PVDF Regular 10 pack 1B4010 01 Continued on next page Accessory Products Continued Additional The following products for use with E PAGE gels are available separately from products Lif2. The staining solutions for the Coomassie R 250 staining protocol and the microwave staining protocol are different Be sure to use the correct stain for the correct protocol For all staining and destaining steps described below be sure to use sufficient reagents to completely cover the gel using a suitable 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 a clean incubation tray Prepare Coomassie stain 0 03 Coomassie R 250 in 30 methanol and 10 acetic acid See note on previous page Stain the gel in the prepared stain for 1 5 hours at room temperature with gentle shaking Destain the gel in destaining solution see previous page at room temperature with gentle shaking with intermittent changes of solution until the bands are visible or overnight for maximum sensitivity and clear background For all staining and destaining steps described below be sure to use sufficient reagents to completely cover the gel in a microwave safe container such that the gel moves freely during the staining and destaining steps 1 10 After electrophoresis remove the gel from the cassette page 17 and place the gel in a clean microwave safe container Prepare Coomassie stain 0 015 Coomassie R 250 in 30 ethanol and 10 acetic acid See note on previous page Add enough stain to completely cover the gel in th
3. Be sure that the well side of the gel is not facing the membrane Reduce methanol concentration in transfer buffer from 15 to 10 if transferring E PAGE 48 Use 0 2 um nitrocellulose membrane for optimal capture of small proteins Use of large pore membranes allow small proteins to blow through Use 10 15 methanol in the transfer buffer No methanol in transfer buffer Use 10 methanol in the transfer buffer No methanol in transfer buffer Appendix Product Specifications E PAGE gel separation range E PAGE 48 Gel specifications The migration and resolution range of proteins run on E PAGE 48 8 Gel E PAGE 48 8 Gel o _ 230 kDa 20 120 kDa 100 kDa x 40 _ 50 kDa 40 to V0 Ooeovr _ 60 T02 g0 Lip Each E PAGE 48 gel contains 48 sample wells and 4 marker wells M Cassette Size Gel Thickness Gel Volume Gel Formulation Well Depth Well Volume Well Opening Running Distance one well to the next Space between Wells 13 5 cm 1 x 10 8 cm w x 0 67 cm thick 3 7 mm 50 mL Proprietary operating at a neutral pH 3mm 15 uL 3 6 mm l x 2 2 mm w 3 2 cm 4 5mm Note E PAGE 48 8 Gels have a unique separation profile which gives protein resolution similar to that of a 4 12 Tris Glycine gel 29 Accessory Products E PAGE Gels Electrophoresis Bases E Holder
4. Gel e Clean staining containers or incubation trays if using the Coomassie R 250 microwave protocol make sure the container is microwave safe Do NOT use the Incubation Tray cat no LC2102 e Rotary shaker For Coomassie R 250 Staining e Coomassie R 250 Stain see note below e Destaining solution 8 acetic acid in deionized water see note below e Methanol regular protocol only e Ethanol microwave protocol only e 2 pieces of nylon membrane microwave protocol only The volume of fixing staining and destaining solutions will depend on the volume of your staining container To obtain good results the volume of solution must be sufficient to cover the gel completely and to allow the gel to move freely during all of the steps When using the Coomassie R 250 microwave staining protocol warm the Coomassie R 250 stain and destaining solution to 50 C without boiling It is important NOT to boil the solutions Since microwave ovens differ significantly we recommend testing various times 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 use these settings for staining 19 Coomassie Staining Continued Note Coomassie R 250 staining protocol Coomassie R 250 staining microwave protocol 20
5. There is no need to remove the E PAGE Gel from the cassette to visualize Lumio fusion proteins 1 Place the gel 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 minimal background as shown on page 25 Coomassie Staining Introduction Materials needed Note Important Instructions for staining E PAGE Gels using Coomassie R 250 are described in this section To obtain maximum sensitivity the total staining time for Coomassie R 250 staining is 1 5 h plus overnight destaining The use of the Coomassie R 250 microwave protocol reduces the amount of time needed for staining and destaining however for minimal background overnight destaining is recommended You will need the following items for staining one E PAGE
6. reconfigures the wells of the E PAGE Gels 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 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 then 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 Use an appropriate gel documentation system to capture a digital image of the gel When imaging the gel should be properly aligned i e not at an angle and gel features should be clear and distinct Proceed to Downloading Software E Editor 2 02 software can be downloaded FREE from our website Go to www lifetechnologies com epage and follow the instructions to download the software and user manual Troubleshooting Troubleshooting The table below provides some solutions to possible problems you might encounter during the electrophoresis of E PAGE Gels No current Daughter E Base Device used Do not use the Daughter E Base Device without a Mother E Base without a Mother E Base Device The Device Daughter E Base Device does
7. routine SDS PAGE and staining or blotting applications Do not use any other SDS PAGE sample buffer See page 7 for more information about loading buffers E PAGE Blotting Pads are supplied with the E PAGE 48 Gels It is necessary to use the pad during semi dry blotting to ensure a good transfer The pad is reusable as long as the pad retains porosity and liquid retaining capacity The E Editor 2 02 software allows you to quickly reconfigure digital images of E PAGE Gel results for analysis and documentation Capture an image of the gel 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 image into other applications for printing saving emailing and or publishing The E Editor 2 02 software can be downloaded FREE at www lifetechnologies epage Follow the instructions to download the software and user manual Methods Guidelines for Sample Preparation Introduction For optimal results using the E PAGE System follow the guidelines for preparing your protein samples as described in this section Prepare your protein samples as described in this section for electrophoresis on E PAGE 48 Gels We recommend that you read this section before preparing your samples Use the sample preparation method and loading buffer appropriate for your detection method Application Loading Buffer Routine
8. side may be facing toward 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 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 from the gel as they can cause air bubbles and field distortion during transfer 22 Semi Dry Blotting of E PAGE Gels Continued Semi Dry blotting protocol continued 6 10 Place the flat side of the gel on top of the blotting membrane such that the well side of the gel is facing up and remove any air bubbles with the Blotting Roller Fill the wells of the gel with 2X NuPAGE Transfer Buffer see page 21 Place the pre soaked E PAGE Blotting Pad on the gel and gently roll out air bubbles using the Blotting Roller Place 2 pieces of 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 with the Blotting Roller Place the cathode plate on the stack without disturbing the blot sandwich Follo
9. staining and 1 page 8 Loading Buffer 1 western blotting Lumio Green Detection 2 page 9 Lumio Gel Sample Buffer Materials needed The following items are needed for sample preparation See page 31 for ordering information 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 page 7 e Optional Lumio Green Detection Kit for detection of Lumio fusion proteins page 9 E PAGE Gels contain SDS and are designed for performing electrophoresis Note under denaturing conditions To obtain the best results perform SDS PAGE under reducing conditions If you need to perform SDS PAGE under non reducing conditions do not add NuPAGE Sample Reducing Agent 10X during sample preparation Guidelines for Sample Preparation Continued Total sample volume Amount of protein High salt or detergent samples Avoid loading less than 5 uL of sample in wells and maintain a uniform loading volume If you do not have enough samples to load all the wells of the gel load an equal volume of deionized water into any empty wells E PAGE Gel Type Recommended Loading Volume E PAGE 48 8 Gel 20 uL We recommend loading 5 10 pL of deionized water into all wells of the E PAGE Gel prior to loading samples or molecular weight markers Load up to 20 ug of protein per well of the E PAGE Gel T
10. 64776 M Copper E PAGE 48 usr sss2702 sses invitrogen electrode E PAGE 48 8 Gels have a unique separation profile which gives protein resolution similar to that of a 4 12 Tris Glycine gel Specifically the following table describes the separation range of the E PAGE 48 8 Gels see page 29 10 Resolution Range 30 Lower Resolution 30 Upper Resolution Range Range 90 80 kDa 10 50 kDa 80 200 kDa E PAGE 48 Gels are used with a specially designed electrophoresis device that combines a base and a power supply Two types of devices are available from Life Technologies e The Mother E Base Device Cat 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 48 Gel Note The Mother E Base Device has been tested for electrophoresis with up to three Daughter E Bases connected at one time e The Daughter E Base Device Cat no EB D03 connects to the Mother E Base and together they can be used for the independent electrophoresis of 2 or more E PAGE 48 Gels Note The Daughter E Base Device does not have an electrical plug and cannot be used without a Mother E Base Device E PAGE Protein Electrophoresis System Continued E PAGE Loading Buffer E PAGE Blotting Pad E Editor 2 02 Software E PAGE Gels are supplied with E PAGE Loading Buffer 1 4X which is optimized for E PAGE Gels when performing
11. E PAGE 48 Experienced Users Procedure ates aie teesciabietaisds fades coos aedcnas aGaa ue io ladaeedodeiunblacianee aeatabiotaenies iv T TOE OR a E A E A A I NE A N E E E TE E rene A 1 AULT OCU CUI OI jieesiin su ceenccna cer esaia raaa aE EEr AEEA SKOS a a inii Aiai ER ai 2 E PAGE Protein Electrophoresis SY StCscdactivnarsaiteesssaalaaiessanticensuanassatiehiasaiteataraebbrar named i 2 INSTI Sacer recreate a 5 Guidelines for Sample Preparations a AA A vate toae 5 Electrophoresis or E PAGE 48 Geleen a ieese earners 10 Guidelines for Loading E PAGE Gels by Automated Liquid Handling ss ssssessesessessestestesessess 15 Maintenance of the E PAGE Devid taso E A A 16 Opening the E PAGE Cassette siaina E cee petiole giant 17 Visualizing and Staining of E PAGE Gels ccccccccsssssesssesssessseesseesseessnesssesssnessneesaeesaes 18 Vistializine Lumio TUSON PrO DS er a A 18 Coomassie Sta MN ar a a E a nen at EE 19 BIOting of E PAGE Gelsin 21 Semi Dry Blottine of EPAGE Cebra a dhacieisicinld uaateeesioeataaiee 21 Expected Resultan aina a Gaeat gs steadiid tastes cie cesta auctnsetst Aa nesath a atahtaneataases 24 Usm E Editor 2202 SORE WAL C semenn ae eas fugas o a Urai E Ta ea 26 Troubles TO OUI sene N E E oe sls Seca raat 27 Appen orn A 29 Product SpecdiNcaNoNS nsession rin E T AE 29 Teehnnical onp Or asians A A E E A OE A R 32 ill E PAGE 48 Experienced Users Procedure Introduction This quick reference sheet is inc
12. USER GUIDE novex vy Life technologies E PAGE 48 Protein Electrophoresis System For electrophoresis of 48 protein samples Catalog Number EP048 08 Publication Part Number 25 0735 Publication Number MAN0000374 Revision A 0 For Research Use Only Not for use in diagnostic procedures d technologies For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF IMPORTANT LICENSING INFORMATION These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses TRADEMARKS 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified ll Contents
13. assette Load the gel There should be a uniform volume of liquid in each well The recommended total volume for each well is 20 uL 1 Load 5 10 pL deionized water to each well of the E PAGE Gel prior to loading your samples or protein molecular weight standard 2 Load 10 5 uL of samples in loading buffer into the wells using a multichannel pipetter or an automated liquid handling system 3 Load the appropriate protein molecular weight standards in the marker wells of the gel See page 7 for recommended molecular weight standards 12 Electrophoresis of E PAGE 48 Gels Continued Perform Note It is not necessary to have a gel in the Mother E Base Device if you are electrophoresis using a Daughter E Base Device However the Mother E Base Device must be with the E Base plugged into an electrical outlet Device 1 Press and release the pwr prg button to begin electrophoresis The red light changes to green and the digital display shows the count down time while the run is in progress Mother E Base Device es Pwr prg On a Daughter E Base Device press and release the pwr prg button on the Daughter E Base Device to begin electrophoresis Mother E Base Device L Daughter E Base Device To increase the run time while the run is in progress press the time button to select the desired time and then release the time button Do not run an E PAGE 48 Gel for more than 30 minutes To in
14. ate the plates Repeat on each side of the cassette until the plates are completely separated Caution Use caution while inserting the Gel Knife between the two plates to avoid excessive pressure on the gel 3 Gently pull apart the cassette halves with your hands until the cassette halves are completely separated and the gel is exposed 4 Carefully remove the gel from the cassette 5 Use the Gel Knife to trim the top and bottom electrode areas of the gel 6 Proceed to blotting page 21 or staining pages 18 Note 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 or 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 Visualizing and Staining of E PAGE Gels Visualizing Lumio Fusion Proteins Introduction Visualizing n TM Lumio fusion proteins 18 The steps involved in detecting Lumio fusion proteins run on an E PAGE Gel are described below To visualize Lumio fusion protein bands after electrophoresis you will need a UV transilluminator or a laser based scanner see below For further details on imaging Lumio fusion proteins refer to the product manual available at www lifetechnologies com or contact Technical Support page 32 After electrophoresis is complete immediately visualize and image the gel as described below
15. e Technologies Product Catalog no SeeBlue Plus2 Pre Stained Standard LC5925 E PAGE SeeBlue Pre stained Protein Standard LC5700 MagicMark XP Western Standard LC5602 E PAGE MagicMark Unstained Protein Standard LC5701 BenchMark Fluorescent Protein Standard LC5928 Lumio Green Detection Kit LC6090 SYPRO Ruby Protein Gel Stain S 12000 SimplyBlue SafeStain LC6060 SilverQuest Silver Staining Kit LC6070 SilverXpress Silver Staining Kit LC6100 InVision His tag In gel Stain LC6030 NuPAGE Transfer Buffer 20X NP0006 NuPAGE Antioxidant NP0005 NuPAGE Sample Reducing Agent 10X NP0009 Nitrocellulose Filter Paper Sandwich 0 45 pm LC2006 Nitrocellulose Filter Paper Sandwich 0 2 um LC2009 Invitrolon PVDF Filter Paper Sandwich 0 45 pm LC2007 Blotting Filter Paper 2 5 mm LC2008 E PAGE Blotting Pad LC2101 Blotting Roller LC2100 Incubation Tray LC2102 Gel Knife E19010 Large Gel Drying Kit NI2207 Gel Dry Drying Solution 1X LC4025 WesternBreeze Chromogenic Kit Anti Mouse 1 kit WB7103 Anti Rabbit 1 kit WB7105 Anti Goat 1 kit WB7107 WesternBreeze Chemiluminescent Kit Anti Mouse 1 kit WB7104 Anti Rabbit 1 kit WB7106 Anti Goat 1 kit WB7108 Pro Q Diamond Phosphoprotein Gel Stain P 33300 Pro Q Diamond Phosphoprotein Gel Destain Solution P 33310 Pro Q Sapphire 532 Oligohistidine Gel Stain P 33354 31 Technical Support Obtaining support Safety Data Sheets SDS Certificate of Analysis Limited warra
16. e microwave safe container Warm the gel and solution to about 50 C in a microwave oven see page 19 Note Do NOT boil the solution Incubate the gel in the warmed staining solution for 30 minutes on an orbital shaker at room temperature Discard the stain rinse the gel briefly with water and discard the water Add enough destaining solution see previous page to cover the gel during incubation Place two pieces of positively charged nylon membrane on top of the destaining solution to speed up the destaining process Warm the destaining solution gel and nylon membrane to 50 C ina microwave oven see previous page Note Do NOT boil the solution 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 Results obtained with Coomassie stain are shown on page 24 Blotting of E PAGE Gels Semi Dry Blotting of E PAGE Gels Introduction Additional blotting protocols Materials needed Preparing 2X transfer buffer A semi dry blotting procedure for blotting E PAGE 48 Gels is described in this section You will need a semi dry apparatus that can accommodate the dimensions of an E PAGE Gel Guidelines for semi wet and other blotting methods can be found in the E PAGE Technical Guide available at www invitrogen com or contact Technical Support page 32 For ordering informatio
17. eeBlue Plus2 Pre stained Standard 5 uL MagicMark XP Western Protein Standard 10 uL Benchmark His tagged Protein Standard 10 pL diluted 1 5 Lysozyme 200 ng Carbonic Anhydrase 200 ng BSA 100 ng E coli lysate 5 uL Expected Results Continued Lumio Green detection Results obtained with an E PAGE 48 8 Gel using the Lumio Green Detection Kit are shown below Various concentrations of a Lumio fusion protein were labeled with Lumio Green Detection Kit and run on an E PAGE 48 8 Gel as described in this manual M 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 E PAGE 48 usp ssez702 506 072 Invitrogen E LACI The gel contains the following samples lanes not indicated are blank Lane M 2 8 13 19 26 32 37 43 3 9 14 20 27 33 38 44 4 10 15 21 28 34 39 45 5 11 16 22 29 35 40 46 6 12 17 23 30 36 41 47 Sample BenchMark Fluorescent Marker 5 uL Human kinase Lumio fusion protein 10 uL E coli CAT Lumio fusion protein 10 uL E coli GUS Lumio fusion protein 10 uL E coli calmodulin Lumio fusion protein 10 pL E coli kinase D Lumio fusion protein 10 uL 25 Using E Editor 2 02 Software Introduction Imaging the gel Downloading software 26 The E Editor 2 02 software for Windows allows you to reconfigure digital images of E PAGE Gels for analysis and documentation E Editor 2 02 software
18. es If you need to load multiple gels on a robotic platform while other gels are running on the E Base Electrophoresis Devices use an E Holder Platform page 15 e Perform manual loading with a pipette or multichannel pipetter load samples into alternate wells of the gel followed by a second round of loading into the remaining wells e Use short rigid tips for loading E PAGE Gels e Always load 5 10 pL deionized water first into all wells prior to loading sample or molecular weight standard e E PAGE Gels can only be used once Do not re use e To obtain the best results run the E PAGE Gel immediately after removal from the pouch and loading e Store and run E PAGE Gels at room temperature e For optimal results do not run 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 e Avoid running samples containing different salt or protein concentrations in adjacent lanes e Avoid touching the gel during electrophoresis Each E PAGE 48 Gel cassette is labeled with an individual barcode The barcode facilitates the identification of each gel cassette during electrophoresis of multiple gels Each E PAGE cassette contains an EAN 39 type of barcode which is recognized by the majority of commercially available barcode reader
19. he amount of protein required depends on the staining or western detection method used for visualization If you are unsure of how much protein to use test a range of concentrations to determine the optimal concentration for your sample 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 pg L of the final sample volume Excess protein will cause poor resolution Samples containing high salt or detergents result in loss of resolution on E PAGE Gels Dilute the samples such that the final concentration of the salt or detergent in the sample is as described below NT not tested Detergent or Salt Final Concentration for E PAGE 48Gels Triton X 100 lt 0 3 Tween 20 lt 0 3 CHAPS lt 0 3 NP 40 lt 0 3 RIPA lt 0 25X SDS lt 2 already in loading buffer Tris lt 300 mM NaCl lt 300 mM Ammonium sulfate lt 100 mM Sodium acetate lt 200 mM EDTA lt 20 mM MES Not recommended DTT Glycine Urea Imidazole No effect seen up to 500 mM Guidelines for Sample Preparation Continued Loading buffer Molecular weight standards Based on your application use the appropriate loading buffer as described below SDS PAGE and routine staining Method 1 page 8 For SDS PAGE and routine staining or blotting use the E PAGE Loading Buffer 1 4X included in the kit for preparing samples The E PAGE Loading Buffer 1 4X is op
20. ied with the following product Product Catalog no E PAGE 48 8 Gels EP048 08 Each pack of E PAGE Gels contain the following contents E PAGE Gel 8 Pack Contents Quantity E PAGE Gels 8 gels E PAGE Loading Buffer 1 4X 4 5 mL E PAGE Blotting Pad 1 The E PAGE 48 and E PAGE Blotting Pad are shipped and stored at room temperature Do not allow the temperature to drop below 4 C or rise above 40 C when storing the gels The E PAGE 48 8 Gels are guaranteed stable for 6 months from the date of production when stored properly The expiration date is printed on the package The E PAGE Loading Buffer is shipped at room temperature Store the E PAGE Loading Buffer 1 4X at room temperature or at 4 C Introduction E PAGE Protein Electrophoresis System About the E PAGE Protein Electrophoresis System Applications E PAGE 48 Gels The E PAGE Protein Electrophoresis System is designed for fast medium to high throughput protein electrophoresis in a horizontal format The E PAGE 48 System consists of the following components E PAGE 48 8 Pre cast Gels E Base Electrophoresis Device E PAGE Loading Buffer 1 4X E PAGE Blotting Pad E Editor 2 02 Software The E PAGE 48 Protein Electrophoresis System is ideal for protein analysis and screening including Coomassie R 250 staining page 19 In gel staining with Lumio Green Reagent page 18 Silver staining and SYPRO Ruby staining techni
21. ientation is available for some automation systems with the use of a 90 adapter If your system does not have an adapter that allows the Portrait configuration please contact Technical Support page 32 to obtain a 90 adapter Loading E PAGE 48 Gels requires one of the following loading patterns depending on your machine Fixed Tip 4 movements per row 8 8 4 4 FEE A srerana hip Min E in Ma se Se ae i t i E Variable Tip 3 movements per row 8 8 8 ee Continued on next page 15 Maintenance of the E PAGE Device Maintaining the E Base Device 16 Keep the surfaces of the Mother E Base Device and Daughter E Base Device 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 Life Technologies Disconnect the Mother E Base Device from the outlet when not in use for a prolonged period of time Opening the E PAGE Cassette Opening the To remove the E PAGE 48 Gel from the cassette for blotting or staining use the E PAGE 48 Gel Knife to open the cassette cassette with the 4 Separate each of the bonded sides of the E PAGE 48 cassette by inserting Gel Knife the Gel Knife into the gap between the two plastic plates that make up the cassette The side of the cassette with the wells should face up 2 Lever the handle of the knife gently to separ
22. 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 with shaking Preparing blotting Nitrocellulose membrane 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 a 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 the membrane in deionized water 3 Soak the membrane in a 2X NuPAGE Transfer Buffer see previous page for several minutes in the Incubation Tray Semi Dry blotting 1 Ina clean container or Incubation Tray soak 4 pieces of 2 5 mm Blotting Filter Paper 8 6 cm x 13 5 cm in 2X NuPAGE Transfer Buffer see page 21 Remove any air protocol bubbles trapped between filter paper 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 see page 21 Press the pad to ensure the elimination of any visible air bubbles Inspect both sides of the pad for air bubbles before use Note The Blotting Pad has no specific orientation either
23. luded for experienced users of E PAGE 48 Gels If you are a first time user follow the detailed protocols provided in this manual Note For optimal results load each E PAGE 48 Gel within 20 minutes of removing the gel from the foil pouch and run within 10 minutes of loading Prepare Sample Use up to 20 ug protein per lane of the E PAGE 48 Gel See page 5 for sample preparation Select Program 1 Plug the Mother E Base into an electrical outlet Connect the Daughter E Base to a and Load Electrophoresis with E Base Opening the E PAGE 48 Cassette Staining E PAGE 48 Gels Using E Editor 2 02 Software 1V Mother E Base or to another Daughter E Base connected to a Mother E Base Select the program EP for E PAGE 48 Gels using the pwr prg button Manually change the run time to 23 minutes using the time button 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 Do not exceed 15 uL of total well volume First Load Deionized Water Then Load Sample in Loading Buffer 5 10 uL 10 5 pL Load the appropriate protein molecular weight marker 5 10 uL in the four marker wells of the gel Press and release the pwr prg button located on the lower right corner of the base to begin electropho
24. ly prior to use 1 Prepare your samples in a total volume of 10 pL in the E PAGE Loading Buffer 1 4X as described below If you need to prepare samples in a volume of 5 15 pL adjust the volumes accordingly Reagent Non reduced a Loading Buffer 1 2 5 uL 2 5 uL NuPAGE Sample 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 Gels page 12 Guidelines for Sample Preparation Continued Method 2 Lumio A brief protocol to prepare samples for specific detection of Lumio fusion detection proteins using the Lumio Green Detection Kit is described below For details on the Lumio Green Detection Kit refer to the manual available at www lifetechnologies com or contact Technical Support page 32 1 Refer to the Lumio Detection manual for details on each type of protein Prepare protein samples as follows Purified sample 7 9 UL 2 9 UL Protein Sample Sample Lumio Gel Sample Buffer 4X Volume Volume Bacterial samples 7 9 UL 2 9 UL Mammalian lysate 7 5 UL Zo UE Partially purified 7 5 uL 2 5 uL sample n vitro expressed 10 uL Not needed There is no need to add Lumio Gel Sample Buffer 4X as the sample is already prepared in this buffer 2 Thaw the Lumio Green Detection Reagent and mix well 3 Add 0 1 pL Lumio Green Detection Reagent to the protein samples from Step 1 in a fume hood Mix well Return
25. n see page 31 e Semi dry blotter e Methanol e NuPAGE Transfer Buffer 20X e NuPAGE Antioxidant e Blotting membranes Invitrolon Filter Paper Sandwich or Nitrocellulose 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 IMPORTANT To ensure a good transfer it is necessary to use the E PAGE Blotting Pad and 2 5 mm Blotting Filter Paper We recommend using 2X NuPAGE Transfer Buffer with 15 methanol and NuPAGE Antioxidant for optimal transfer of most proteins from E PAGE Gels When the complete transfer of higher molecular weight proteins gt 150 kDa is desired reduce methanol to 10 For one gel prepare 500 mL of 2X NuPAGE Transfer Buffer Buffer Component 2X NuPAGE Transfer 2X NuPAGE Transfer Buffer with 15 Methanol Buffer with 10 Methanol NuPAGE 50 mL 50 mL Transfer Buffer 20X NuPAGE 0 5 mL 0 5 mL co to 500 mL to 500 mL 21 Semi Dry Blotting of E PAGE Gels Continued Equilibrating the Equilibration of the gel in transfer buffer results in the removal of salts that may gel increase conductivity and heat during transfer Perform equilibration for the recommended time as longer equilibration results 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
26. not have an electrical plug to connect to an electrical outlet No electric contact no Copper contacts in the base are Make sure that the copper contacts in the base red light when damaged due to improper use are intact oo is inserted or Expired or defective gel cassette Use properly stored gels before the specified tee not start no used expiration date green light E PAGE cassette is not Remove cassette and reinsert When the correctly inserted into base cassette is correctly inserted and power is on a fan in the base begins to run and a steady red light illuminates on the base page 13 Sample leaking from Sample is overloaded or wells Be sure to load the recommended volume of the wells are damaged sample per well page 12 Remove the comb carefully without damaging the wells Poor resolution or Sample is overloaded Do not load more than 20 ug of protein smearing of bands sample per well Very low volumes of sample Do not load less than 5 pL of sample Always were loaded load 10 20 uL deionized water in all wells prior to sample loading For proper band separation we recommend keeping sample volumes uniform and loading deionized water into empty wells Incorrect loading buffer used Make sure that protein sample is in one of Use the recommended loading buffers as described on page 7 Electrophoresis was not started For best results the gel should be run within immediately after sample 15 minute
27. nty 32 For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches Safety Data Sheets SDSs are available at www lifetechnologies com support The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Life Technologies and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on the Life Technologies web site at http www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies For support visit www lifetechnologies com support or email techsupport dlifetech com www lifetechnologies com technologies 29 October 2014
28. ques protocols available in the E PAGE Technical Guide Western blotting Functional assays IMPORTANT E PAGE 48 Gel are not compatible with the E Gel 96 base 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 E PAGE 48 Gels are self contained pre cast gels that include a buffered gel matrix and electrodes packaged inside a disposable UV transparent cassette Each E PAGE 48 Gel contains 48 sample lanes and 4 marker lanes This configuration provides a 3 2 cm run length The wells of E PAGE 48 Gels are compatible for loading with a multichannel pipetter in alternating lanes or with an automated liquid handling system see page 15 for automation specifications After electrophoresis the E PAGE 48 cassette is easily opened with a Gel Knife to remove the gel for staining or blotting applications Each E PAGE 48 cassette is labeled with an individual barcode to facilitate identification of the gel using commercial barcode readers page 10 See page 29 for E PAGE 48 8 Gel specifications E PAGE Protein Electrophoresis System Continued Diagram of an E PAGE 48 Cassette Separation range About the E Base System A diagram of the E PAGE 48 Gel cassette is shown below ee electrode Plastic combs Barcode M 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 4
29. resis 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 Press and release the pwr prg button to stop the beeping Remove the E PAGE 48 cassette from the base Open the gel cassette for staining or blotting applications Open the E PAGE 48 cassette with a Gel Knife to remove the gel Insert the Gel Knife in the gap between the two plastic plates of the cassette and lever at each of the four edges to separate the two halves Pull apart the cassette halves with your hands until the cassette halves are separated Using the Gel Knife trim the top and bottom electrode areas of the gel Proceed to staining or blotting Stain E PAGE 48 Gels using Coomassie R 250 stain page 19 or other staining methods detailed information in the E PAGE Technical Guide available for downloading from www lifetechnolgies com or by contacting Technical Support page 32 For blotting E PAGE 48 Gels see page 21 Use an appropriate digital documentation system to capture a digital image of the gel Download E Editor 2 02 software and the instruction manual for free at www lifetechnolgies com epage Use the E Editor 2 02 software to align and arrange the lanes in the image and save the reconfigured image for further analysis Product Contents Types of kits Kit contents Shipping and storage This manual is suppl
30. s Refer to the manufacturer s instructions to set up the barcode reader Note When capturing an image of an E PAGE 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 Electrophoresis of E PAGE 48 Gels Continued Select a program Use program EP for running E PAGE 48 Gels Select the program prior to on the E Base inserting a gel into the base Device Brief instructions for using the E Base Device are included in this manual For details refer to the E Base manual 1 Plug Mother E Base Device into an electrical outlet using the electrical plug on the base Connect a Daughter E Base Device to a Mother E Base Device or another Daughter E Base Device connected to a Mother E Base Device The display shows EP or last program used EG or EP 3 Press and release the pwr prg button to select program EP 4 Press and release the time button to view the time setting The default run time for program EP is 14 minutes 5 Press and hold the time button to increase the time to 25 minutes If the time button is not released the time setting increases until it reaches 00 To begin cycling through the numbers again starting from 00 press the time button again Do not run E PAGE 48 Gels for more than 30 minutes e When performing electrophoresis of Lumio fusion proteins extend the No
31. s of sample loading loading Continued on next page 2I Troubleshooting Continued Observation Poor resolution or smearing of bands Over run the gel or need more time to run gel Protein bands 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 Uneven transfer of proteins and edge lanes during semi dry blotting of E PAGE gt 48 Gel Weak transfer of proteins during semi wet blotting 28 Solution Cause High salt or detergent concentration Be sure the final concentration of salt or in samples detergent in the sample is as described on page 6 You may need to manually increase the run time for high salt or detergent samples to obtain optimal results Use properly stored gels before the specified expiration date Select program EP for E PAGE Gels If you accidentally selected an incorrect program and are at the beginning of the run stop the run and select the desired program Expired gel used Accidentally selected an incorrect 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 Be sure that the E PAGE Blotting Pad is used correctly Non uniform electric field created around wells Incorrect gel orientation Not enough SDS in sample
32. se the pwr prg button The light 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 as described on page 11 and rerun the gel In case of an external power failure loss of electricity or the electrical cord is accidentally removed from the outlet the run will continue when the power resumes The Mother E Base or Daughter E Base Device signals 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 had occurred during the run Guidelines for Loading E PAGE Gels by Automated Liquid Handling Automated loading of E PAGE 48 Gels Important Loading patterns for E PAGE 48 Gels E PAGE 48 Gels are compatible with any automated liquid handling system that has an 8 span loading head with either fixed 9 mm or variable distance between the loading heads These loading patterns are described below To download programming scripts for your automated liquid handling system go to www lifetechnologies com epage For automated loading of E PAGE 48 Gels position the plate in the Portrait orientation rather than the Landscape position as shown below Portrait Landscape This or
33. te run time of the gel for an additional 2 minutes to prevent the formation of a fluorescent dye front e If your sample contains high salt or detergent concentrations you may need to manually increase the run time e To increase the run time when a cassette is inserted press and release the time button to increase the time setting by 1 minute intervals or press and hold the time button to increase the time continuously e To increase the run time while a run is in progress see next page To manually interrupt or stop a run see page 14 1 Electrophoresis of E PAGE 48 Gels Continued Insert the gel 1 Open the package and remove the gel cassette 2 Remove the plastic combs from the gel Slide the gel into the Mother E Base or Daughter E Base Device The two copper electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base as shown below Cathode oe i Electrode lt a raar ere TE N Anode Electrode ban S Digital i _ Pwr prg display pi ime When the gel is properly inserted into the base a fan in the base begins running and a red light illuminates at the lower left corner of the base The digital display shows the appropriate time for a selected program or the last time setting Ready Mode Note If a cassette is inserted into the base before selecting program EP remove the cassette select program EP and reinsert the c
34. terrupt or stop a run in progress see page 14 13 Electrophoresis of E PAGE 48 Gels Continued Perform electrophoresis with the E Base Device continued Interrupting electrophoresis 14 2 The Mother E Base or Daughter E Base Device signals the end of the run with a flashing red light and an audible alarm that beeps rapidly for 2 minutes followed by a single beep every minute The digital display shows the original time setting it does not indicate time changes that were made during electrophoresis The digital display also shows the elapsed time since the end of the run up to 19 minutes 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 or Daughter E Base Device 5 For visualizing Lumio fusion proteins see page 18 For opening the E PAGE cassette see page 17 Note The bands in the gel will diffuse within 40 minutes 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 was interrupted You can remove the gel from the E Base Device 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 relea
35. the Lumio Green Detection Reagent to 20 C immediately after use Incubate the samples at 70 C for 10 minutes 5 Allow samples to cool for 1 2 minutes and centrifuge briefly at maximum speed in a microcentrifuge 6 Thaw the Lumio In Gel Detection Enhancer and mix well Add 1 uL of Lumio In Gel Detection Enhancer to the samples 7 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 8 Proceed to Loading E PAGE Gels page 10 When performing electrophoresis of Lumio fusion proteins extending the run Note time of the gel for an additional 2 minutes can prevent the formation of a fluorescent dye front Electrophoresis of E PAGE 48 Gels Introduction General guidelines Using the barcode 10 After preparing your samples you are ready to load E PAGE Gels This section describes the procedure for loading protein samples and molecular weight standards The Mother E Base and Daughter E Base Electrophoresis Devices are designed to fit most robotic platforms allowing you to load and run E PAGE Gels directly on the automated liquid handling system 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 48 Gel This ensures proper loading of samples into the wells See page 15 for automation guidelin
36. timized for E PAGE Gels Do not use any other types of SDS PAGE sample buffer SDS PAGE and detection of Lumio Fusion Proteins Method 2 page 9 For in gel detection of Lumio fusion proteins with the Lumio Green Detection Kit use the Lumio Gel Sample Buffer 4X included with the Lumio Green Detection Kit This buffer is specifically formulated to provide optimal results with the Lumio Green Detection Reagent Do not use the E PAGE Loading Buffer 1 4X or any other type of SDS PAGE buffer to prepare samples for Lumio Green detection The following protein molecular weight standards and loading volumes are recommended for E PAGE Gels Because molecular weight standards have different migration patterns in different gel systems see page 24 for apparent molecular weight calibration on E PAGE Gels See page 31 for ordering information E PAGE 48 SeeBlue Plus2 Pre 5 ul Sa eee 8 Gel stained Standard H P MagicMark XP l Electrophoresis Western Protein x i followed by staining Standard MagicMark XP Western Protein Western blotting Standard BenchMark Fluorescent Fluorescent Protein l Detection Standard Guidelines for Sample Preparation Continued Method 1 Routine Use this protocol if you are performing SDS PAGE followed by routine staining staining and or blotting blotting If the E PAGE Loading Buffer 1 4X is stored at 4 C bring the buffer to room temperature and mix brief
37. w the manufacturer s instructions to further assemble the semi dry blotting apparatus Transfer at 25 V for 1 h 19 V cm You may need to optimize the transfer conditions for your specific proteins or semi dry blot apparatus 29 Expected Results Molecular weight calibration Results with Coomassie R 250 staining 24 Apparent molecular weight values for SeeBlue Plus2 Pre stained Standard on an E PAGE 48 8 Gel are shown below Myosin 215 kDa Phosphorylase 146 kDa as BSA 78 kDa Glutamic Dehydrogenase 56 kDa Alcohol Dehydrogenase 39 kDa Carbonic Anhydrase 25 kDa Myoglobin Red 19 kDa Lysozyme 16 kDa Samples were run on an E PAGE 48 8 Gel and stained with Coomassie R 250 using the microwave staining protocol as described in this manual page 20 Results after staining are shown below M 1 2 3 45 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M p m b err IOE ment ai l l os M E5 OESTRA Pr Hiv wb atia b aril ear a a ni ee Ses _ _ _ T O png ma Lame o _ i a g y a 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 M The gel contains the following samples rows not indicated are blank Lane M 2 4 21 23 26 28 45 47 6 19 30 45 8 32 10 34 12 36 15 17 39 41 Sample S
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