iBlot™ Gel Transfer Device - Ecologie Microbienne Lyon
Contents
1. Discard the iBlot Anode stack Bottom At this point the iBlot Gel Transfer Device is ready for another run no cooling period is required If you are not using the device turn off the power switch located on the back of the iBlot Gel Transfer Device Important Do not reuse the iBlot Disposable Sponge iBlot Filter Paper and iBlot Cathode and Anode Stacks after blotting Discard after each use Cleaning and Clean the blotting surface Metal Spacers 1 and 2 and the De bubbling Roller Maintenance with a damp cloth or paper tissue Allow the parts to dry before use For any other repairs and service contact Technical Support page 33 Do not perform any repairs or service on the iBlot Gel Transfer device to avoid damaging the iBlot Device 24 Post Transfer Analysis and Optimizing Blotting Post Transfer Analysis Optimizing Blotting After the transfer you may proceed to immunodetection store the membrane for future use or stain the membrane For immunodetection of proteins use the WesternBreeze Chromogenic or Chemiluminescent Immunodetection Kits available from Invitrogen page x or any other immunodetection kit For storing nitrocellulose membranes air dry the membrane and store the membrane in an air tight plastic bag at room temperature or 4 C Avoid storing nitrocellulose below 20 C as they turn brittle For storing PVDF membranes air dry the membrane and store the membrane in a2. Assembling the iBlot Device continued 20 Place the prerun gel on the transfer membrane of the anode stack as described e One midi gel on an iBlot Gel Transfer Stack e Two mini gels head to head on an iBlot Gel Transfer Stack figure A e One mini gel on an iBlot Gel Transfer Stack Mini figure B A B In a clean container soak one iBlot Filter Paper or Mini Filter paper based on the gel type used in deionized water iBlot Filter Paper is included with each iBlot Gel Transfer Stacks Place the presoaked iBlot Filter Paper on the prerun gel Use the Blotting Roller to remove any air bubbles between the membrane and gel as shown below for the Transfer Stack For E PAGE gels there is no need to use a filter paper and be sure to use the Blotting Roller over the well rows to flatten any remaining gel protrusions to ensure even transfer 7 Remove the iBlot Cathode Stack Top or Cathode Stack Mini from the package Discard the red plastic tray Continued on next page Using the iBlot Device with the Blotting Roller Continued Assembling the iBlot Device continued Performing Blotting 8 Place the iBlot Cathode Stack Top or Cathode Stack Mini on top of the presoaked filter paper with the copper electrode side facing up and aligned to the right of the bottom stack Remove any air bubbles using the Blotting Roller Place the iBlot Disposable Sponge on
3. Button Metal Spacer 1 Blotting Surface The blotting surface is the area where the iBlot Gel Transfer Stacks are placed with the gel to perform blotting This area also contains the Gel Barriers that guide the proper placement of the transfer stacks to allow correct electrical contact De Bubbling Surface The de bubbling surface is the area where de bubbling of E PAGE gels is performed using the De bubbling Roller This area contains Metal Spacers 1 and 2 and hinges to attach the De bubbling Roller Barriers are also present on the de bubbling surface to guide the proper placement of the iBlot Anode Stack Bottom and the gel to allow efficient de bubbling The iBlot Anode Stack Bottom is assembled with the gel Metal Spacers 1 and 2 the iBlot Cathode Stack Top and the De bubbling Roller The entire assembled transfer stack with the gel is pulled together with the pull tab towards the blotting surface resulting in removal of any trapped air bubbles between the gel and the blotting membrane Continued on next page Description of Parts Continued iBlot Gel Transfer Device continued Programs Lid The iBlot Lid contains ventilation holes to allow for proper ventilation of the unit during the run The iBlot Disposable Sponge page 7 is placed on the inner side of the iBlot Lid within the small protrusions present on the lid that allow proper placement of the sponge The Lid also contains the electrica
4. Continued Assembling the iBlot Device continued Performing Blotting 11 12 Hold the iBlot E PAGE Tab and plastic tab on the iBlot Anode Stack Bottom together and pull the assembly anode and cathode stacks and gel together through the De bubbling Roller towards the blotting surface in one smooth uninterrupted movement until the assembly reaches the Gel Barriers on the blotting surface figure A At the end of de bubbling all layers are aligned to the right as shown below figure B A B Place the iBlot Disposable Sponge on the inner side of the lid between the small protrusions on the lid that hold the sponge in its place such that the metal contact is to the top right The sponge absorbs any excess liquid generated during blotting and exerts an even pressure on the stack surface After assembling the iBlot Gel Transfer Device perform blotting within 15 minutes of assembling the stacks with the gel as described below 1 Close the iBlot Lid and secure the latch The red light is on indicating a closed circuit Ensure the correct program is selected page 12 Press the Start Stop button to start the transfer The red status light changes to green The transfer continues using the programmed parameters At the end of the transfer current automatically shuts off and the iBlot Gel Transfer Device signals the end of transfer with repeated beeping sounds and flashing red light and di
5. Copper electrode eS Transfer Gel Layer The iBlot Cathode Stack is available in standard format for blotting E PAGE midi or two mini gels see page vii for dimensions and Mini format for blotting one mini gel Dispose the iBlot Cathode Stack Top after every use Do not reuse the iBlot Cathode Stack Top Do not use the iBlot Cathode Stack Top with the tray in the iBlot Device The iBlot Disposable Sponge is placed on the inner side of the iBlot Lid within the small protrusions on the lid The iBlot Disposable Sponge absorbs any excess liquid on the stacks formed during blotting and generates an even pressure on the stack assembly See page vii for dimensions of the iBlot Disposable Sponge Metal contact The iBlot Disposable Sponge is comprised of white melamine and an aluminum metal contact The metal contact is fixed onto the sponge at a distance of 15 mm from the upper right corner The metal contact allows proper contact with the electrical contact on the lid as well as the electrode on the assembled iBlot Gel Transfer Stacks Discard the iBlot Disposable Sponge after every use Do not reuse the iBlot Disposable Sponge The iBlot Filter Paper is used for blotting mini or midi gels The iBlot Filter Paper is placed on top of the thin gel before placing the iBlot Cathode Stack Top to protect the gel integrity during the blotting process The iBlot Filter Paper is sup
6. Proprietary Copper 14 1 cm 1 x 8 5 cm w x 0 32 cm thick 8 5 cm 1 x 8 5 cm w x 0 32 cm thick Proprietary Copper Nitrocellulose 0 2 um or PVDF 0 2 pm low fluorescence 16 8 cm x 10 3 cm 1 7 cm wide copper contact 15 cm 1 x 9 5 cm w White Melamine Aluminum 13 5 cm 1 x 8 cm 1 x 0 04 cm thick 8 cm 1 x 8 cm w x 0 04 cm thick vii iBlot Gel Transfer Device Front View of The front top view showing various parts of the iBlot Gel Transfer Device is iBlot Device shown below Metal Spacers De bubbling 1 and 2 Roller Start Stop Button Rear View of The rear view showing various parts of the iBlot Gel Transfer Device is shown iBlot Device below Power Switch USB Port AC inlet Continued on next page viii iBlot Gel Transfer Device Continued Control Panel of The control panel of the iBlot Gel Transfer Device is described below iBlot Device The Digital Display shows six digits that specify the transfer conditions as follows e First two digits indicate the program name e Remaining four digits specify the time of transfer in minutes and seconds respectively The Select Button is used to toggle between program and time The Up Down Buttons are used to increase or decrease the program or time See page 12 for details on selecting the program Digital Select Up Down Display Button Buttons 1X Accessory Products iBlot Gel Transfer iBl
7. assembling the device with iBlot Gel Transfer Stacks and your gel 1 When the electrophoresis of your samples is almost complete press the power switch located on the rear of the device page viii to turn ON the iBlot Gel Transfer Device The fan in the device begins to run and digital display shows text which is stabilized in few seconds to display the default parameters P 3 0 7 00 or last program used Select the appropriate program based on the gel type by pressing the Select button to toggle between program minutes and seconds Once the selected item blinks use the Up Down Buttons for changing the values to the desired parameters as shown below Gel Type Program Volts Run Time E PAGE 48 P3 20 7 8 minutes E PAGE 96 P3 20 7 8 minutes Novex Midi Gel 1 mm thick P3 20 7 8 minutes 2 Mini Gels 1 0 or 1 5mm thick P3 20 7 8 minutes 1 Mini Gel 1 0 or 1 5 mm thick P3 20 7 8 minutes using mini transfer stacks Custom parameters are also easily created using a combination of programs P1 P5 and time up to the time limit listed for each gel type page 5 for a specific gel type not listed above Note You may need to optimize the blotting parameters volts or time based on your initial results See page 25 for optimizing blotting conditions Note The maximum voltage and current of the output to gel stacks is 25 VDC and 5 5 Amp 12 Using the iBlot Device with the De Bubbling Roller Introduction Ma
8. briefly pressing Start Stop button or restart iBlot Anode Stack the run by pressing and holding the Bottom Start Stop button The layers are not aligned Align the layers properly as described in the protocols Ensure that the electrodes are in contact Current is above 5 5 amp Select a program with a lower voltage Open the iBlot Lid and ensure the stacks are aligned properly Close the lid and restart the run by subtracting the time already elapsed Replace the iBlot Gel Transfer Stacks with fresh transfer stacks Ensure an iBlot Filter Paper was used during blotting of mini or midi gels Digital display shows The layers are not aligned Align the layers properly as described in the Error3 indicating a protocols Ensure the iBlot Anode Stack partial short circuit Bottom tray is aligned to the Gel barriers on right edge of the blotting surface to avoid any accidental contact of the lid electrical contacts with the iBlot Anode Stack Bottom Corrosion of the iBlot Incorrect placement of the top Be sure the iBlot Cathode Stack Top is placed Cathode Stack Top stack correctly with the copper electrode side facing up Avoid placing the top stack in the inverted position No proteins No current or incorrect See previous page to ensure the electrical transferred to the program used circuit is complete and current is flowing membrane through the device Be sure to use the correct program page 12 Empty
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10. spots on the Presence of air bubbles e Be sure to remove all air bubbles between membrane between the gel and the the gel and membrane by using the De membrane preventing the Bubbling Roller for E PAGE Gels or transfer of proteins Blotting Roller for other gels Expired or creased e Use the iBlot Gel Transfer Stacks before membranes used the expiration date printed on the package Continued on next page 30 Troubleshooting Continued Protein bands distorted on membrane for E PAGE gels High molecular weight proteins remain in the gel indicated by staining of the gel after transfer Membrane and the gel turns blue iBlot Anode Stack Bottom transfer gel melts to a viscous blue solution Signal intensity is similar for different protein loads after detection Non uniform electric field created around wells Incorrect program or transfer conditions used Note It is normal for some proteins to remain in the gel as some high molecular weight proteins do not transfer completely using the iBlot Gel Transfer Device as compared to semi wet transfer apparatus Longer transfer times result in the deposition of copper ions Membrane is trimmed to fit the gel size resulting in direct contact between the iBlot Cathode Top and Anode Bottom stacks High protein load detection is not within the linear range Ensure that the well protrusions on the E PAGE gel are flattened properly using the
11. the gel adhered to the other plate For details on removing the gel refer to the manual supplied with the mini or midi gel For other gel types refer to the manufacturer s recommendations to remove the gel from the cassette There is no need for any pretreatment of the gel after electrophoresis The transfer membrane is supplied in a ready to use format in the stacks without any need for pretreatment Do not treat the PVDF membrane with methanol as the PVDF membrane is preactivated prior to assembly with the transfer stack You may blot E PAGE gels using the blotting protocol with the Blotting Roller If you wish to use the Blotting Roller for blotting E PAGE gels be sure to e Wash the E PAGE gel briefly in deionized water prior to blotting to remove any small gel pieces attached to the gel e Use the Blotting Roller all over the gel including all well areas to obtain efficient blotting If you notice distorted protein bands after using the E PAGE blotting protocol with the Blotting Roller we recommend that you blot the E PAGE gels using the De bubbling Roller page 13 Continued on next page Using the iBlot Device with the Blotting Roller Continued Use the appropriate iBlot Gel Transfer Stacks based on the gel that you are Important blotting Do not trim the membrane or transfer stacks to fit the size of your gel as the transfer quality is not affected if the prerun gel is smaller than the transf
12. the inner side of the lid between the small protrusions on the lid that hold the sponge in its place such that the metal contact is to the top right as shown below The sponge absorbs any excess liquid generated during blotting and exerts an even pressure on the stack surface After assembling the iBlot Gel Transfer Device perform blotting as described below Perform blotting within 15 minutes of assembling the stacks with the gel 1 Close the iBlot Lid and secure the latch The red light is on indicating a closed circuit Ensure that the correct program is selected page 12 Press the Start Stop button to start the transfer The red status light changes to green The transfer continues using the programmed parameters page 12 At the end of the transfer current automatically shuts off and the iBlot Gel Transfer Device signals the end of transfer with repeated beeping sounds and flashing red light and digital display Note Previous versions of the iBlot Gel Transfer Device firmware versions prior to 2 7 9 signaled the end of transfer with repeated beeping sounds and flashing green light instead of red light and digital display Press and release the Start Stop button to stop the beeping The light turns to a steady red light Proceed to Disassembling the iBlot Gel Transfer Device page 24 21 iBlot Quick Reference Guide Introduction A quick reference guide for operating the iBlot Gel Transfer Device
13. using the following criteria Visual Inspection Each component of the iBlot Gel Transfer Stack is visually inspected as follows Transfer Gel Layer is inspected for size integrity uniformity absence of bubbles and spots and must meet the set specifications Copper Electrodes must be clean without any blue or other spots Plastic Tray must be clean without any deformations and the electrode contact attached to the bottom plastic tray must be proper without showing any sign of corrosion Nitrocellulose and PVDF Membrane must be clean and free of spots or bubbles and must not be dry Functional Test The iBlot Dry Blotting System is functionally qualified by blotting suitable E PAGE and NuPAGE gels as described in this manual using pre stained protein standards and protein samples The system is tested for initial and final current on the device detection sensitivity and the presence of bubbles smears distortions after blotting on the membrane and must meet the set specifications Purchaser Notification Limited Use Label License No 5 Invitrogen Technology 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 pr
14. Breeze Chromogenic Anti Mouse Kit page x using 1 10 000 dilution of an anti tubulin and 1 5 000 anti actin antibody Samples on the gel Lanes 1 10 5 ul SeeBlue Plus2 Pre Stained Protein Standard Lanes 2 7 SW480 lysate 0 5 ug pl 0 125 ul 0 25 ul 0 5 ul 1 pl 2 pl 4 ul respectively Lanes 8 9 11 12 MagicMark XP Western Protein Standard 0 5 pl 1 pl 2 yl 4 ul respectively 12345 6789 10 1112 Tubulin iis en Actin a o ik a gt 7 _ Troubleshooting Introduction Review the information below to troubleshoot your experiments using the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks To troubleshoot immunodetection refer to the manufacturer s recommendations for optimizing immunodetection No current red light is Incomplete electric circuit not on after securing due to the lid iBlot Disposable Sponge covers the metal contact or the metal contact on the sponge is on the left Incorrect position of the transfer stacks or improper assembly of the transfer stacks Incorrect position of the pull tab iBlot Anode Stack Bottom placed on the device without the tray including the electrical contact iBlot Cathode Stack Top placed on the device with the tray The metal safety contacts in the lid hinge may be dirty and do not make contact Reinsert the iBlot Disposable Sponge such that the metal contact on the
15. CC rules and the European Community Safety requirements Operation of the iBlot Gel Transfer Device is subject to the following conditions e Indoor use e Altitude below 2 000 meters e Temperature range 5 to 40 C e Maximum relative humidity 80 e Installation categories over voltage categories II Pollution degree 2 e Mains supply voltage fluctuations not to exceed 10 of the nominal voltage 100 240 V 50 60 Hz 3 3 A e Mains plug is a disconnect device and must be easily accessible e Do not attempt to open the iBlot Gel Transfer Device To honor the warranty iBlot device can only be opened and serviced by Invitrogen e The protection provided by the equipment may be impaired if the equipment is used in a manner not specified by Invitrogen e The device must be connected to a mains socket outlet with protective earthing connections e Ventilation requirements room ventilation The iBlot Gel Transfer Device complies with part 15 of the FCC rules Operation of the device is subject to the following two conditions e The device may not cause harmful interference e The device must accept any interference received including interference that may cause undesired operation Ethrog Biotechnologies Ltd an Invitrogen company is the manufacturer and owner of the UL file For more information contact Ethrog Biotechnologies Ltd Ness Ziona Science Park Bldg 22 P O Box 4035 Ness Ziona Israel 74103 The
16. Caution symbol denotes a risk of safety hazard Refer to accompanying documentation WEEE Waste Electrical and Electronic Equipment symbol Technical Support Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds 33 Product Qualification Product Qualification 34 The iBlot Gel Transfer Stacks are qualified
17. De bubbling Roller or Blotting Roller To ensure best blotting results we recommend using the De bubbling Roller with E PAGE gels If you used the Blotting Roller with E PAGE gels be sure to follow the recommendations on page 18 to obtain good results Use the appropriate program and run time based on the gel type as described on page 12 For mini or midi gels e Usea lower gel percentage to separate the high molecular weight proteins Increase the transfer time in 30 second increments Perform an equilibration step as described on page 25 to improve transfer For E PAGE gels e Increase the transfer time in 30 second increments e Use P2 program for 8 minutes Be sure to perform the transfer for the recommended time for each gel type Always maintain the membrane size identical to the transfer stack Transfer quality is not affected by smaller gel size compared to the membrane Since the immunodetection sensitivity is higher for dry blotting with iBlot device when compared to semi dry or wet blotting we recommend that you decrease the protein load use more diluted antibody or perform detection for shorter time You may need to perform some optimization based on your initial results 31 Appendix Explanation of Symbols and Warnings CE LISTED FE A Caution WEEE The iBlot Gel Transfer Device complies with the Underwriters Laboratories Inc regulation part 15 of the F
18. Instructions are provided below to assemble the iBlot Gel Transfer Device iBlot Device with iBlot Gel Transfer Stacks and E PAGE precast gels See page 18 for blotting mini midi or other gels 1 Open the lid of the device and pull up the Metal Spacers 1 and 2 If you have attached the De bubbling Roller to the device then remove the roller as shown in the figure below 2 Remove the package labeled iBlot Anode Stack Bottom from the iBlot Gel Transfer Stacks Box Remove the laminated sealing of the iBlot Anode Stack Bottom and keep the stack in the transparent plastic tray Mhe l 3 Place the iBlot Anode Stack Bottom stack with the tray to the left of the blotting surface area such that the tab on the tray is on the right side of the De bubbling Roller as shown below Slide the bottom stack to the left until the stack is blocked by the Gel Barriers present on the left side of the device Note Always handle the iBlot Anode Stack Bottom using the plastic tray without disturbing the gel and membrane layers in the stack Do not touch the transfer membrane on the stack Continued on next page 14 Using the iBlot Device with the De Bubbling Roller Continued Assembling the iBlot Device continued Clean the Metal Spacer 1 with a damp cloth or tissue and place the spacer on the membrane as shown below Place the prerun gel containing your protein samples on Metal Spacer 1 such that the g
19. Invitrogen iBlot Dry Blotting System For dry electroblotting of proteins from mini midi and E PAGE gels Version C 7 February 2007 25 0911 ii Table of Contents TADIS OF COMUCINS ieiei5 Gs eSecssica stata T stasalea acecgueecelets iii Prodiict Contei snit e anaana Ena vV Product SpeciNcaN ONS S vi Blot Gelliansier Devi ceereretr ener em ore eer e a rer ene ee ere ee re viii PNCCESSOLY FP HOCUICIS aene E ems acl E T ala eeenae ee oe aeaa eat Xx ANE O CQUIC ONY we sinicisaissiswasleacaiacalendatdacialecattatintuarca EEEE EENAA EE EEE A 1 CNV CIV TOW coeire A area ess a A a ataaneah e a TA R 1 TSS ETI E a autouatarmag E E A N N alameeg reassess 4 Experimenta Ov Cre Wages a T R eee emia 9 IV CUNO CS a E E saaaididetaunweea eens cant vansneas canaaageumnacnenbanacase tas 10 General CUI IM Sroine n E T te qusssmecnscueelesteacecgteecne en 10 Cettin otare desanat a a aa 12 Using the iBlot Device with the De Bubbling Roller eee eeseeeeseeseseeeeseeesseeesseeessesesseeesaees 13 Using the iBlot Device with the Blotting Roller 0 0 cssssssssssssscssssessssesssecssssesesesseseesesessssesseseeeees 18 IBO Quick Rererence i CS saren Sapir aeca et Van each ayaa ave coc 22 Disassembling the iBlot Gel Transiter DOV 1Ce coke satecnssatauthosdonentna sense tassonevas acest AEE RAEE E Eana AiE 24 Post Transfer Analysis and Optimizing Blotting y ssc saccscinsatisaioasecsratiedeiotatedtoail einen Aashanetactatisenidates 25 Examp lesot RESUS ai
20. aa E a E casa agesttatbanpuata shtWashiteditel tt oamahitbass 27 TTOUDIES TO OUINS aroni n a eects nea A teen uaa Ate E cadat 29 716 01 196 Gera arr ne sient ene eines se nS ee tea ae Sena er Rar et eee a eee 32 Explanation Of Symbols and Waring S sssrinin eaea anaia ERE AE IA TE O Taaa anisa 32 Techne k UP OOM suan N r E a S 33 Product QualificatiO Mesran cha aseanaeiaddeies ca vacadoinataks amb nerdehe aa ceevaeaeniaaes 34 Purchaser Nonticatn orenen ai E N A meaqecauecneiass 35 Warrant y orenen a na 36 iii 1V Product Contents Types of Products This manual is supplied with the following products iBlot Gel Transfer Device IB1001 IB1001UK IB1001EU iBlot Gel The contents of the iBlot Gel Transfer Device are listed below Transfer Device Component Quantity Contents iBlot Gel Transfer Device 1 each Specific Power Cord based on the type of unit ordered 1 each for U S Canada Taiwan Japan Europe or UK Blotting Roller 1 each De bubbling Roller 1 each iBlot E PAGE Tab 10 See page vi for specifications and description of the iBlot Gel Transfer Device Upon Receiving Examine the unit carefully for any damage incurred during transit File any the Instrument damage claims with the carrier The warranty does not cover in transit damage iBlot Transfer The following iBlot Transfer Stacks are available from Invitrogen Stacks Product Transfer Membrane Catalog No iBlot Gel Transfer Stacks Regular Nitro
21. air tight plastic bag at room temperature 4 C or 80 C When you are ready to use the membrane rewet the membrane with methanol for a few seconds followed by thorough rinsing of the membrane with deionized water to remove methanol For staining membranes after blotting you may use any total protein membrane staining methods such as Coomassie Blue R 250 Ponceau S Amido Black or SYPRO Ruby Blot Stain page x The iBlot Gel Transfer Device blotting protocol is compatible with most total protein membrane staining methods listed above Note The sensitivity of total protein membrane staining after using the dry blotting protocol with iBlot Gel Transfer Device is slightly lower than the total membrane protein staining obtained with the semi wet transfer protocol However due to the nature of dry blotting lower transfer does not affect the immunodetection sensitivity If you do not detect any proteins on the membrane after immunodetection or staining refer to Troubleshooting on page 29 Refer to the manufacturer s recommendations for optimizing immunodetection When using the iBlot Gel Transfer Device most proteins transfer efficiently using the protocol in this manual Based on specific properties of a protein or a set of proteins some optimization of the blotting protocol may be necessary Perform optimization of blotting as follows Performing an equilibration step prior to transfer To improve the transfer of h
22. and appropriate cathode and anode buffers in the gel matrix to allow fast reliable dry blotting of proteins without the need to prepare buffers See page 6 for details Important features of the iBlot Dry Blotting System are listed below e User friendly iBlot Gel Transfer Device design with an integrated power supply for fast reliable protein transfer within 7 minutes e Ability to perform blotting of E PAGE mini and midi gels e Unique iBlot Gel Transfer Stacks with integrated nitrocellulose or PVDF transfer membrane allow dry electroblotting of proteins without the need to prepare buffers and are compatible for use with NuPAGE Bis Tris and Tris Acetate Tris Glycine Tricine and E PAGE gels e Pre programmed iBlot Gel Transfer Device with 5 programs for transfer of proteins from various gel types e Dry blotting enables higher immunodetection sensitivity e Built in safety features in the device enhance user safety Continued on next page Overview Continued System Overview The iBlot Dry Blotting System is based on the dry blotting concept which utilizes the unique patented gel matrix technology developed by Invitrogen for E Gel and E PAGE gels To use the iBlot Dry Blotting System for protein transfer assemble the iBlot Gel Transfer Stacks containing the nitrocellulose or PVDF transfer membrane with your pre electrophoresed gel on the iBlot Gel Transfer Device Any trapped air bu
23. bbles that interfere with efficient protein transfer are removed using the De bubbling Roller for E PAGE gels or using the Blotting Roller for mini or midi gels The blotting is performed using a specific program The following features of the iBlot Dry Blotting System allow rapid protein transfer without the need for external power supply or premade buffers The iBlot Gel Transfer Stacks act as ion reservoirs that contain the appropriate anode and cathode buffers incorporated into the gel matrix eliminating the need for premade buffers or soaked filter papers and minimizing handling resulting in consistent performance The iBlot Gel Transfer Stacks also contain the copper electrodes anode and cathode required for electrophoresis The protein transfer consistency is increased since the copper anode does not generate oxygen gas as a result of water electrolysis as compared to conventional inert electrodes present in other blotting systems See figure below The design of the iBlot Gel Transfer Device reduces the distance between electrodes and the integrated power supply This unique design combined with the gel matrix technology of the iBlot Gel Transfer Stacks allows the system to generate high field strength and high protein currents increasing the transfer speed Schematic of iBlot Dry Blotting System showing the flow of current Copper Cathode te 2H 03H 70H iBlot Cathode Stack Top Pre run g
24. cellulose IB3010 01 PVDF IB4010 01 iBlot Gel Transfer Stacks Mini Nitrocellulose IB3010 02 PVDF IB4010 02 If you ordered the iBlot Gel Transfer Stacks Regular or Mini you will receive the components listed in the table below Store the iBlot Gel Transfer Stacks at room temperature For best results use the transfer stack before the expiration date printed on the package for each stack aie The iBlot Gel Transfer Stacks Regular contain iBlot Cathode Stack Top iBlot Anode Stack Bottom iBlot Disposable Sponge iBlot Filter Paper iBlot E PAGE Tab The iBlot Gel Transfer Stacks Mini contain iBlot Cathode Stack Top iBlot Anode Stack Bottom iBlot Disposable Sponge iBlot Filter Paper Mini Product Specifications iBlot Gel Transfer Device Specifications vi Dimensions Weight Electrical Parameters Built in Features Compatibility iBlot Materials Operating Temperature Blotting Roller 37 cm 1 x 20 cm w x 11 cm h 2 3 kg 100 240 V 50 60 Hz 3 3 A Digital display alarm light LED Suitable for transfer of mini 8 x 8 cm midi 8 x 13 cm and E PAGE Gels Polycarbonate Cycoloy Acrylic Gold plated copper Stainless steel Plasticized silicone Aluminum 5 40 C Delrin roller attached to a stainless steel handle 8 6 cm wide The iBlot Gel Transfer Device is impervious to alcohol acid HCI alkali NaOH but not co
25. cture 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 Email outlicensing invitrogen com 35 Warranty iBlot Gel Transfer Device 36 Invitrogen warrants that this product will be free from defects in material and workmanship for a period of one 1 year from date of purchase If a defect is present Invitrogen will at its option repair replace or refund the purchase price of this product at no charge to you provided it is returned during the warranty period This warranty does not apply if the product has been damaged by accident abuse misuse or misapplication or from ordinary wear and tear For your protection items being returned must be insured against possible damage or loss This warranty shall be limited to the replacement of defective products It is expressly agreed that this warranty will be in lieu of all warranties of fitness and in lieu of the warranty of merchantability 2006 2007 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
26. el Blotting membrane iBlot Anode Stack Bottom PERE EEE EH SSES Continued on next page Overview Continued Transfer Membrane Purpose of the Manual Downloading Upgrades The iBlot Gel Transfer Stacks are assembled with the transfer membrane and are available with Nitrocellulose membrane 0 2 um The nitrocellulose membrane is composed of 100 pure nitrocellulose to provide high quality transfer The membrane is compatible with commonly used detection methods such as staining immunodetection fluorescence or radiolabeling but not recommended for reprobing The proteins bind to the membrane due to hydrophobic and electrostatic interactions The protein binding capacity is 209 ug cm PVDF membrane 0 2 um low fluorescence The PVDF membrane has higher binding capacity than nitrocellulose and is physically stronger than nitrocellulose allowing reprobing of proteins The PVDF membrane is preactivated and ready for use without any pretreatment with alcohols The membrane is compatible with commonly used detection methods such as staining immunodetection fluorescence or radiolabeling The proteins bind to the membrane due to hydrophobic interactions The protein binding capacity is 240 ug cm This manual provides the following information Overview of the dry blotting process to transfer proteins Details and specifications on the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks Protocol
27. el is aligned to the lower right corner of the bottom stack with the wells of the E PAGE gel facing up Clean the Metal Spacer 2 with a damp cloth or tissue and place the spacer over the gel as shown below Remove the package labeled iBlot Cathode Stack Top from the iBlot Gel Transfer Stacks Box Remove the iBlot Cathode Stack Top from the package Continued on next page 15 Using the iBlot Device with the De Bubbling Roller Continued Assembling the 8 Insert the steel iBlot E PAGE Tab in the plastic tray groove with the tab iBlot Device teeth facing up figure A Gently press the iBlot Cathode Stack over the continued teeth to allow the teeth to penetrate into the copper electrode figure B Remove the iBlot Cathode Stack Top from the red plastic tray using the iBlot E PAGE Tab figure C 9 Place the iBlot Cathode Stack Top without the tray on top of Metal Spacer 2 with the copper electrode side facing up Ensure that all layers are aligned to the right to perform efficient de bubbling 10 Insert the De bubbling Roller into the two grooves and lower the roller to its lowest location while holding the pull tab The resulting assembly consists of the gel and cathode and anode stacks placed between two Metal Spacers 1 and 2 with the De bubbling Roller on top of the assembly as shown below Continued on next page 16 Using the iBlot Device with the De Bubbling Roller
28. er stack Always maintain the membrane size identical to the transfer stacks to avoid accidental contact between the iBlot Anode and Cathode Stacks See page 10 for gel types compatible with the iBlot Gel Transfer Device e Use the iBlot Gel Transfer Stacks Regular for blotting two mini gels or one midi gel e Use the iBlot Gel Transfer Stacks Mini for blotting one mini gel Assembling the Instructions are provided below to assemble the iBlot Gel Transfer Device iBlot Device with iBlot Gel Transfer Stacks or Mini and mini midi or other gels See page 13 for blotting E PAGE gels 1 Open the lid of the iBlot Gel Transfer Device Ensure the blotting surface is clean 2 Remove the iBlot Anode Stack Bottom or Mini stack from the package Remove the laminated sealing of the iBlot Anode Stack Bottom and keep the stack in the transparent plastic tray Place the iBlot Anode Stack Bottom with the tray directly on the blotting surface under the round lid Align the anode stack to the Gel barriers on right edge of the blotting surface see figure below to avoid accidental contact of the electrical contacts on lid with the iBlot Anode Stack Bottom 3 Ensure no bubbles are visible between the membrane and the transfer stack gel below the membrane Remove any trapped air bubbles using the Blotting Roller Continued on next page 19 Using the iBlot Device with the Blotting Roller Continued
29. gital display Note Previous versions of the iBlot Gel Transfer Device firmware versions prior to 2 7 9 signaled the end of transfer with repeated beeping sounds and flashing green light instead of red light and digital display Press and release the Start Stop button to stop the beeping The light turns to a steady red light Proceed to Disassembling the iBlot Gel Transfer Device page 24 17 Using the iBlot Device with the Blotting Roller Introduction Instructions are provided in this section to assemble the iBlot Gel Transfer Device without the De Bubbling Roller for blotting mini midi or other gels If you wish to blot E PAGE gels see page 13 for the blotting protocol Materials Needed You will need the following items Prerun mini or midi gel containing your protein samples and standards iBlot Gel Transfer Stacks for blotting one midi gel or two mini gels page x iBlot Gel Transfer Stacks Mini for blotting one mini gel page x Blotting Roller supplied with the device Removing the Gel Remove the gel from the cassette for transfer after completion of electrophoresis as described below Note 18 Open the mini or midi gel cassette using the Gel Knife by inserting the knife into the narrow gap between the two plates of the cassette Push up and down gently on the knife s handle to separate the plates Upon opening the cassette discard the plate without the gel and slowly remove
30. iBlot Gel Types Transfer Stacks are listed below Note The iBlot Gel Transfer Device and iBlot Gel Transfer Stack is not yet optimized for Northern or Southern blotting with agarose gels E Gel or TBE polyacrylamide gels Gel Type iBlot Gel Transfer Stack E PAGE 48 or 96 Gels 13 5 cm 1 x 10 8 cm w iBlot Gel Transfer Stack 3 7 mm thick Regular Midi Gels 13 cm 1 x 8 3 cm w iBlot Gel Transfer Stack NuPAGE Novex Bis Tris Tris Acetate or 1 0 mm thick Regular Tris Glycine Midi Gels or equivalent gels Mini Gels 8 cm 1 x 8 cm w iBlot Gel Transfer Stack NuPAGE Bis Tris or Tris Acetate Tricine 1 0 and 1 5 mm thick Regular or iBlot Gel Tris Glycine Gels or equivalent gels Transfer Stack Mini Recommended Use the following parameters for blotting based on the gel type that you use Parameters Gel Type Program Volts Run Time E PAGE 48 P3 20 7 8 minutes E PAGE 96 P3 20 7 8 minutes Novex Midi Gel 1 mm thick P3 20 7 8 minutes 2 Mini Gels 1 0 or 1 5 mm thick P3 20 7 8 minutes 1 Mini Gel 1 0 or 1 5 mm thick P3 20 7 8 minutes using mini transfer stacks Custom parameters are also easily created using a combination of programs P1 P5 and time up to the time limit listed for each program for gel types not listed above Continued on next page 10 General Guidelines Continued Recommended Protocols Note Use the following recommended blotting protocols based o
31. ibody gt main Ir Two NuPAGE Novex 4 12 Bis Tris Mini Gels were subjected to blotting using the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks with the Blotting Roller as described in this manual The proteins on the nitrocellulose membrane were detected using the WesternBreeze Chromogenic Anti Rabbit Kit page x using 1 2 000 dilution of an anti E coli antibody Lane 1 5 ul SeeBlue Plus2 Pre Stained Protein Standard Lanes 2 9 Duplicate samples of E coli lysate diluted 1 16 0 5 ul 1 ul 2 ul 4 pl respectively tier maiia HL N j i i DaN non Ip i T i pi v ih pai j l J Iacono f 27 Expected Results Continued Mini Gel Results Using Nitrocellulose Mini Gel Results Using PVDF 28 SeeBlue Plus2 Pre Stained Protein Standard 5 pl was electrophoresed on a NuPAGE Novex 4 12 Bis Tris Mini Gel After electrophoresis the gel was subjected to blotting using the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks Mini with Blotting Roller as described in this manual The figure below shows good transfer of protein standard bands on to the nitrocellulose membrane ww cee ces cee Ge ee ae Ge The NuPAGE Novex 4 12 Bis Tris Mini Gel was subjected to blotting using the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks with the Blotting Roller as described in this manual The proteins on the PVDF membrane were detected using the Western
32. igh molecular weight proteins from mini or midi NuPAGE or Tris Glycine gels equilibrate the gel in 100 ml Equilibration Buffer 2X NuPAGE Transfer Buffer containing 10 methanol and 1 1000 NuPAGE Antioxidant for 20 minutes at room temperature on a shaker prior to transfer After equilibration use the gel for transfer using the iBlot Device as described in this manual Note The equilibration step improves the transfer of high molecular weight proteins but may increase the blow through of low molecular weight proteins Do not use this equilibration step with E PAGE gels as no improvement in the transfer is observed Increasing or decreasing the transfer time Based on the initial results you can increase or decrease the transfer time using the Up Down buttons in 30 second increments Do not perform transfer for more than the time limit indicated for each program page 5 Continued on next page 25 Post Transfer Analysis and Optimizing Blotting Continued 26 Note It is normal for some proteins to remain in the gel as some high molecular weight proteins do not transfer completely using the iBlot Gel Transfer Device as compared to wet transfer apparatus Since the sensitivity of detection using the iBlot Gel Transfer Device is higher as compared to semi wet and semi dry blotting complete transfer of proteins is not required Almost complete transfer of prestained standard protein bands is observed with the iBl
33. is provided below Display iBlot connected to an Version of iBlot electrical outlet and power firmware switch is on iBlot No transfer stacks detected Default setting or the turned on with lid opened last defined program time appear Program Press and release the Select Program name blinks selection Button Time Press the Select Button and Minutes blink selection use the Up and Down minutes Buttons to change values Time Press the Select Button and Seconds blink selection use the Up and Down seconds Buttons to change values Ready to Transfer stacks placed in Steady red Program and time run the device and lid closed Run Press and release the Steady green Count down time Start Stop button Running Open the lid and fix the Continuous Flashing red Error1 Error2 or error alert error lost contact with beeping Error3 stacks or short circuit Error Close the lid Continuous Flashing Errorl Error2 or fixed beeping ereen Error3 Continue Press and release the Steady green Count down time after error Start Stop button Restart Press and hold the after error Start Stop button e Transfer stacks placed in e Steady red the device and lid closed No transfer stacks detected with lid opened Error 3 is displayed in iBlot Gel Transfer Device with firmware version 2 8 1 and above e Program and time e Default setting or the last defined program time appear Continued on
34. l contacts for the copper electrodes on the stack to complete the electrical circuit Start Stop Button The Start Stop Button is located near the blotting surface and is used to activate the run stop the run or reset the program The red and green status light indicates the status of the run or errors Control Panel The Control Panel is located near the de bubbling surface and contains the 6 digit digital display Select button and Up Down Buttons See page ix for control panel details The iBlot Gel Transfer Device is pre programmed with 5 voltage programs that allow blotting using a different combination of volts and time The 5 programs are listed in the table below Default Run Time Run Time Limit The Default Run Time is the default time shown for each program which can be increased or decreased using the Up Down Buttons see page 12 The Run Time Limit is the maximum run time that can be programmed for the specific program See page 12 to select an appropriate program based on the gel type Continued on next page Description of Parts Continued iBlot Anode Stack Bottom The iBlot Anode Stack Bottom package contains a copper electrode nitrocellulose 0 2 um or PVDF 0 2 um membrane and the Bottom Transfer Gel Layer packaged in a transparent plastic tray The transparent plastic tray serves as the support for assembling the transfer stacks with the gel and has a tab that assists in the mo
35. mpatible with acetone dimethyl sulfoxide and acetic acid The CE mark symbolizes that the product conforms to all applicable European Community provisions for which this marking is required The iBlot Gel Transfer Device complies with the Underwriters Laboratories Inc regulation and the European Community Safety requirements Operation of the iBlot Gel Transfer Device is subject to the conditions described in this manual The protection provided by the equipment may be impaired if the equipment is used in a manner not specified by Invitrogen Continued on next page Product Specifications Continued iBlot Gel Transfer Stack Specifications The iBlot Gel Transfer Stacks are used with the iBlot Gel Transfer Device and are available separately from Invitrogen page x For details on the iBlot Transfer Stacks see page 6 The specifications for the iBlot Transfer Stacks are listed below iBlot Cathode Stack Top Top Stack Gel Layer Regular Top Stack Gel Layer Mini Top Stack Gel Layer Composition Electrode iBlot Anode Stack Bottom Bottom Stack Gel Layer Regular Bottom Stack Gel Layer Mini Bottom Stack Gel Layer Composition Electrode Transfer Membrane Plastic Tray iBlot Disposable Sponge Dimensions Material Metal Contact iBlot Filter Paper Regular Filter Paper Mini Filter Paper 13 6 cm 1 x 8 5 cm w x 0 19 cm thick 8 5 cm 1 x 8 5 cm w x 0 19 cm thick
36. n the gel type that you use e For E PAGE gels use the blotting protocol with the De bubbling Roller described on page 13 e For mini or midi gels use the blotting protocol with the Blotting Roller described on page 18 To obtain the best results follow these recommendations e Wear gloves at all times during the entire blotting procedure to prevent contamination of gels and membranes e Do not touch the membrane or gel with bare or gloved hands This may contaminate the gel or membrane and interfere with further analysis If you need to adjust the membrane always use forceps e Use the appropriate gel type and iBlot Gel Transfer Stacks as described on the previous page e Avoid using expired iBlot Gel Transfer Stacks Always use the transfer stacks before the specified expiration date printed on the package e Remove air bubbles as indicated in the protocol using the De bubbler Roller or Blotting Roller supplied with the device e Do not trim the membrane or iBlot Gel Transfer Stacks to fit your gel size See previous page for gel sizes that are compatible with iBlot Device Note that iBlot Gel Transfer Stacks Mini are available for blotting mini gels page x Maintain the membrane size identical to the transfer stacks to avoid direct contact between the top and bottom transfer stacks We have observed increased immunodetection sensitivity with the iBlot Dry Blotting System If you are using the iBlot sy
37. next page 22 iBlot Quick Reference Guide Continued End of run Automatic Continuous beeping Flashing red Program and time for 2 minutes or less if Start Stop button is pressed followed by a single beep every minute Run ends Transfer stacks placed in Steady red Program and time after an the device and lid closed external power failure 23 Disassembling the iBlot Gel Transfer Device Introduction Refer to the instructions below to disassemble the iBlot Gel Transfer Device Procedure To obtain good transfer and detection results disassemble the device and stacks within 30 minutes of ending the blotting procedure 1 Open the lid of the iBlot Device 2 Remove the iBlot E PAGE Tab used for blotting E PAGE gels only Rinse the tab with deionized water and store in a dry place for future use Do not discard the iBlot E PAGE Tab 3 Discard the iBlot Disposable Sponge and iBlot Cathode Stack Top 4 Carefully remove and discard the gel and filter paper if used as shown below Remove the transfer membrane from the stack and proceed with the blocking procedure or stain the membrane see next page for details Note If you are using PVDF membranes place the membrane immediately into the blocking or staining solution or water as PVDF membranes dry quickly If the PVDF membrane is dried rewet the membrane with methanol and rinse with deionized water a few times before use
38. oduct 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 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 patents owned or controlled by Invitrogen Corporation which cover this product 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 manufa
39. ot Gel Transfer Device However note that the complete transfer of prestained protein standards does not indicate complete transfer of other proteins Examples of Results Introduction E PAGE Gel Results Using Nitrocellulose BSA 66 kDa 2 Two Mini Gel Results Using Nitrocellulose Examples of results obtained using the iBlot Dry Blotting System are shown below E PAGE 48 8 Gel was subjected to blotting using the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks with the De bubbling Roller as described in this manual The proteins on the nitrocellulose membrane were detected using the WesternBreeze Chemiluminescent Anti Mouse Kit page x using 1 10 000 dilution of anti BSA antibody left panel or 1 10 000 dilution of anti tubulin antibody right panel The gel contains the following samples rows not indicated are blank Lane Sample 2 3 4 5 6 and 26 27 28 29 30 BSA 5 ng 10 ng 25 ng 50 ng and 100 ng 8 9 10 11 and 14 15 16 17 MagicMark XP Western Protein 32 33 34 35 and 38 39 40 41 Standard 0 5 ul 1 ul 2 ul and 4 ul 19 20 21 22 23 and 43 44 45 46 47 Human Colon Cancer cell lysate SW480 0 25 ul 0 5 ul 1 ul 2 ul and 4 pl M 1 23 45 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2324M _ T S Tubulin 55 kDa ij k a M 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48M Anti BSA Antibody Anti Tubulin Ant
40. ot Gel Transfer Stacks are available separately from Invitrogen Ordering Stack information is provided below iBlot Gel Transfer Stack Nitrocellulose Regular 1 pack of 10 IB3010 01 iBlot Gel Transfer Stack PVDF Regular 1 pack of 10 IB4010 01 iBlot Gel Transfer Stack Nitrocellulose Mini 1 pack of 10 IB3010 02 iBlot Gel Transfer Stack PVDF Mini 1 pack of 10 IB4010 02 Additional Additional reagents that may be used for electrophoresis of proteins are Products available separately from Invitrogen Ordering information is provided below For more information visit www invitrogen com or call Technical Support page 33 NuPAGE Transfer Buffer 20X NP0006 1 NuPAGE Antioxidant NP0005 WesternBreeze Chromogenic Kit Anti Mouse WB7103 WesternBreeze Chromogenic Kit Anti Rabbit WB7105 WesternBreeze Chemiluminescent Kit Anti Mouse WB7104 WesternBreeze Chemiluminescent Kit Anti Rabbit WB 7106 Blotting Roller LC2100 SeeBlue Plus2 Pre Stained Standard LC5925 MagicMark XP Western Protein Standard LC5602 SYPRO Ruby Protein Blot Stain S 11791 Precast Gels and A large variety of precast gels including NuPAGE Novex Tris Glycine mini Premade Buffers and midi gels and E PAGE gels as well as premade buffers is available from Invitrogen For details contact Technical Support page 33 or visit www invitrogen com Overview Introduction System Components Features Introd
41. plied in two sizes see page vii for dimensions for efficient blotting of mini and midi gels Do not use the iBlot Filter Paper for blotting E PAGE gels Note Failure to use the iBlot Filter Paper during blotting of mini or midi gels may result in high currents exceeding the current limit leading to an error Error2 during the run Continued on next page Description of Parts Continued iBlot E PAGE The iBlot E PAGE Tab is a steel tab used during blotting of E PAGE gels Tab The iBlot E PAGE Tab is attached to the iBlot Cathode Stack Top and is used to pull the transfer stack assembly towards the blotting surface during the de bubbling process of E PAGE gels De Bubbling The De bubbling Roller is a stainless steel aluminum roller designed to remove Roller any air bubbles between the gel and blotting membrane during the assembly of the stacks and gel for blotting E PAGE gels The De bubbling Roller is installed into the hinges on the de bubbling surface The iBlot Gel Transfer Stacks and gel are aligned between the Metal Spacers 1 and 2 and the De bubbling Roller is placed on top The entire gel assembly is pulled together with the pull tab towards the blotting surface to efficiently remove any air bubbles Use the protocol on page 13 to perform blotting using the De Bubbling Roller Do not use the De bubbling Roller for mini midi or gels other than E PAGE gels as these gels may stre
42. sponge is on the top right of the lid and is in contact with the electrode on the transfer stack page 17 Make sure the transfer stack is placed in the proper position in the blotting surface to allow proper contacts with the electrodes Ensure the transfer stacks are assembled correctly use the iBlot Anode Stack Bottom first followed by the gel and iBlot Cathode Stack Top Ensure the pull tab from the iBlot Cathode Stack Top is towards the right of the assembly in the blotting surface page 17 Do not remove the iBlot Anode Stack Bottom from the tray during the assembly The blotting is performed with the bottom stack in the plastic tray Always remove the iBlot Cathode Stack Top from the red plastic tray before placing the top stack on the assembly Do not use the iBlot Cathode Stack Top with the tray Clean the metal safety contacts in the lid hinge with a cotton swab and water Digital display shows The lid opened during the Error1 indicating an run open electrical circuit during the run Close the lid Continue the run by briefly pressing Start Stop button or restart the run by pressing and holding the Start Stop button Continued on next page 29 Troubleshooting Continued Digital display shows The iBlot Cathode Stack Open the lid and align the iBlot Cathode Error2 indicating a Top is touching the Stack Top to the right Continue the run by short circuit copper electrode on the
43. stem for the first time you may need to load less protein use more diluted antibody for detection or perform detection for a shorter time as compared to traditional semi dry or wet blotting systems You may also need to optimize the immunodetection based on your initial results 11 Getting Started Installing the L iBlot Gel Transfer Device pA ce Check the Power Cord supplied with the unit to ensure that the cord is compatible with the local socket format Place the iBlot Gel Transfer Device on a level laboratory bench Keep the area around the device clear to ensure proper ventilation of the unit For your safety Position the device properly such that the power switch and the AC inlet located on the rear of the unit page viii are easily accessible Ensure the AC power switch is in the Off position page viii Attach the power cord to the AC inlet and then to the electrical outlet Use only properly grounded AC outlets and power cords You are ready to use the iBlot Gel Transfer Device for blotting applications See pages 13 18 for the blotting procedure Using the iBlot gt If you are using the iBlot Gel Transfer Device for the first time you may wish Device for the to clean the Metal Spacers 1 and 2 De bubbling Roller and blotting surface First Time with a damp cloth before use Allow the parts to dry before blotting Selecting a You need to select an appropriate program on the iBlot Device prior to Program
44. tch and tear E Blotting Roller The Blotting Roller is a Delrin roller attached to a stainless steel handle 8 6 cm wide The Blotting Roller is used to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks and gel Use the protocol on page 18 to perform blotting of gels using the Blotting Roller Experimental Overview Experimental The table below outlines the experimental steps necessary to perform western Outline blotting using the iBlot Gel Transfer Device For more details on each step see indicated pages Remove the gel from the gel cassette Assemble the iBlot Gel Transfer Device with the iBlot Gel Transfer Stacks and your protein gel using e De bubbling Roller e Blotting Roller Perform western blotting using the recommended parameters Disassemble the iBlot Gel Transfer Device Materials Needed You need the following items Ordering information is on page x e iBlot Gel Transfer Stack for blotting E PAGE Novex Midi Gels or two mini gels see page 10 for recommended gel types e iBlot Gel Transfer Stacks Mini for blotting one mini gel e Prerun gel containing protein samples and protein standards Methods General Guidelines Introduction General guidelines for using the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks are discussed below Recommended Gel The gel types compatible for use with iBlot Gel Transfer Device and
45. terials Needed Removing the Gel Note Instructions are provided in this section to assemble the iBlot Gel Transfer Device with the De Bubbling Roller for blotting E PAGE Gels If you wish to blot mini midi or other gels see page 18 for the blotting protocol You will need the following items e Pre run E PAGE gel or equivalent containing your protein samples and standards e iBlot Gel Transfer Stacks page x Remove the gel from the cassette for transfer after completion of electrophoresis as described below Open the E PAGE cassette using the red plastic Butterfly Opener supplied with the gel to remove the E PAGE gel For details refer to the E PAGE manual supplied with the gel e There is no need for any pretreatment of the gel after electrophoresis Wash the E PAGE gel briefly in deionized water to remove any small gel pieces attached to the gel e The transfer membrane is supplied in a ready to use format in the stacks without any need for pretreatment Do not treat the PVDF membrane with methanol as the PVDF membrane is preactivated prior to assembly with the transfer stack e To obtain the best blotting results with the E PAGE gels we recommend that you use the De bubbling Roller However you may use the Blotting Roller for de bubbling E PAGE gels as described on page 18 Continued on next page 13 Using the iBlot Device with the De Bubbling Roller Continued Assembling the
46. to perform blotting using the iBlot Gel Transfer Device with the De bubbling Roller or Blotting Roller Disassembling the iBlot Gel Transfer Device Tips on optimizing blotting Examples of expected results Troubleshooting Note Immunodetection protocols are not included in this manual Upgrades for the iBlot Device firmware are available To download iBlot Device firmware upgrades go to www invitrogen com iblot Follow instructions on the page to download the upgrades Description of Parts Introduction iBlot Gel Transfer Device The various parts of the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks are described below The iBlot Gel Transfer Device is a blotting device with an integrated power supply capable of producing currents up to 5 5 amp at 25 V Four printed circuit boards hold the electronic components required to process the systems logic unit modify voltage and currents for display and power the blotting process A pre installed firmware controls the parameters such as voltage and time and allows selection of programs see next page for details on each program See page viii for a front and rear view of the device A top view of an open iBlot Gel Transfer Device identifying various parts is shown below Electrical Contact Electrical Contact Metal Spacer 2 a Lid with vents Gel Barriers De bubbli x Gel Surface Barriers t 4 7 gt Start Stop
47. uction The iBlot Dry Blotting System consists of the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks that allows you to quickly and reliably perform western blotting of proteins from various types of gels without the need to prepare buffers The unique design of the iBlot Gel Transfer Device with an integrated power supply combined with the patented gel matrix technology of the iBlot Gel Transfer Stacks generates high field strengths to allow for fast dry blotting of proteins within 7 8 minutes There is no need for an external power supply or to prepare buffers thereby resulting in consistent performance The proteins transferred using the iBlot Dry Blotting System exhibit higher immunodetection sensitivity when compared to proteins transferred using conventional semi dry or semi wet blotting methods See next page to understand how the iBlot Dry Blotting System works and page 4 for details on various parts of the system The iBlot Dry Blotting System consists of e iBlot Gel Transfer Device The iBlot Gel Transfer Device is a self contained blotting unit with an integrated power supply that allows for fast dry blotting of proteins See page 4 for details e iBlot Gel Transfer Stacks The iBlot Gel Transfer Stacks are disposable stacks with an integrated nitrocellulose or PVDF transfer membrane to perform dry blotting of proteins Each iBlot Gel Transfer Stack contains a copper electrode
48. vement of the transfer stack assembly towards the blotting surface during the de bubbling process The Bottom Transfer Gel Layer acts as an ion reservoir and is composed of an optimized proprietary gel composition to provide high quality transfer of proteins within 7 minutes The nitrocellulose 0 2 um and PVDF 0 2 um membranes do not require any pretreatment before use and minimizes protein blow through during the iBlot blotting process Always use the iBlot Anode Stack Bottom with the tray in the iBlot Device See page 7 for iBlot Cathode Stack specifications Transfer membrane Transfer Gel Layer Copper electrode Plastic Tray The iBlot Anode Stack Bottom is available in standard format for blotting E PAGE midi or two mini gels see page vii for dimensions and Mini format for blotting one mini gel Dispose the iBlot Anode Stack Bottom after every use Do not reuse the iBlot Anode Stack Bottom Continued on next page Description of Parts Continued iBlot Cathode Stack Top iBlot Disposable Sponge iBlot Filter Paper The iBlot Cathode Stack Top package contains a copper electrode and the Top Transfer Gel Layer packaged in a red plastic tray The Top Transfer Gel Layer acts as an ion reservoir and is composed of an optimized proprietary gel composition to provide high quality transfer within 7 minutes See page vii for iBlot Cathode Stack specifications 7
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