- Bio-Rad
Contents
1. esssssseeeeeeneeenneeneennnns 21 4 2 Optmizing DNA and RNA Transfer 23 Section 5 Choice of Blotting Membranes eren 23 5 1 Protein Blotting Membranes eseeeeeeneneneenennen 23 5 2 RNA Blotting Membranes eeeee 24 Section 6 Troubleshooting 25 Section 7 2 ee 28 Section 8 Product Information cerent 29 Section 9 References nre tree iri epsas idi tM n ac 30 Section 10 Warran y m 31 Copyright 2002 Bio Rad Laboratories Inc All rights reserved Section 1 General Information 1 1 Introduction The Trans Blot Plus cell is an electrophoretic transfer cell designed for use with large format gels such as those used with the PROTEAN Plus Dodeca cell and for high throughput blotting applications with smaller format gels such as those used with the Criterion Dodeca cell The Trans Blot Plus cell is supplied with three gel holder cassettes each with an effective blotting area of 26 5 x 28 cm A pair of plate electrodes a platinum coated titanium anode and a stainless steel cathode may be positioned 4 cm 7 cm or 10 cm apart for electrophoretic transfers of one two and three gel containing cassettes respectively This variable placement ensures a minimum distance between electrodes which increases the field strength and2. 11 Place the hinge of the upper cassette plate red into the hinge mechanism of the lower plate and lower the upper cassette plate on top of the gel sandwich Make sure that the gel sandwich is aligned below the rim of the hinge so that the cassette will close properly 12 Working with one side at a time apply firm pressure to the area adjacent to a clamp and slide the clamp in 13 Once the cassette is closed and locked insert it into the tank with the hinge side up Make sure the red cassette plate faces the red electrode plate see Figure 4 MAX LEVEL MIN LEVEL Trans Blot Plus Cell 1 CASSETTE 2 CASSETTES 3 CASSETTES Fig 4 2 5 Beginning Transfer 1 Once all the cassettes are in place check that the buffer level is between the maximum and minimum levels indicated on the tank Turn on the stir plate and check that the stirring and cooling are working properly Place the lid on the tank Note The color coded cables on the lid MUST attach to the electrode cards of the same color Reversing the orientation of the cables will cause irreversible damage to the plate electrodes 4 Connect the Trans Blot Plus cell to the power supply Begin the run See Section 3 1 for suggested run times with various buffers 5 Upon completion of the run remove the cassettes and disassemble the gel sandwich on a flat surface so that one locking clamp can be released at a time 2 6 Draining
3. 112 195 1981 Erickson P G Minier L N and Lasher P S J Immun Meth 51 241 1982 Szewcyzyk B and Kozloff L M Anal Biochem 150 403 1985 Perides G Plagens U and Traub P Anal Biochem 152 94 1986 Gershoni J M and Palade G E Anal Biochem 124 396 1982 Gershoni J M and Palade G E Anal Biochem 131 1 1983 Peudelhuber T L Ball D J Davis A H and Garrard W J Nuc Acids Res 10 1311 1982 Towbin H Staehelin T and Gordon J Proc Nat Acad Sci 76 4350 1970 9 Anderson N L Nance S L Pearson T W and Anderson N G Electrophoresis 3 135 1982 Howe J G and Hershey J W B J Biol Chem 2566 12836 1981 Lin W and Kasamatsu H Anal Biochem 128 302 1983 Polvino W J Saravis A Sampson E and Cook B Electrophoresis 4 368 1983 Bio Rad Technical Bulletin 1110 Zeta Probe Blotting Membranes 1982 Coomassie is a trademark of ICI 30 Section 10 Warranty The Trans Blot Plus cell electrophoretic transfer cell is warranted for one 1 year against defects in materials and workmanship If any defects occur during this warranty period Bio Rad Laboratories will repair or replace the defective parts without charge The following defects however are specifically excluded 1 Defects caused by improper operation 2 Repair or modification done by anyone other than Bio Rad Laboratories
4. PLATE ELECTRODE DISTANCE 4cm 7cm 10cm Buffer Power Run Power Run Power Run conditions Time conditions Time conditions Time Towbin 60V 2A 45 min 100V 2A 45 min 100V 5A 60min 90V 3A 15 30 120V 2 4A 15 30 min 130V 2 3A 15 30 min CAPS 41V 2A 15 min 66V 2A 30 min 95V 2A 30 min 60V 3A 15 min 100V 3A 15 30 min 120V 2 5A 15 30 min Carbonate 25V 2A 15 min 40V 2A 30 min 55V 2A 30 min 35V 3A 15 min 55V 3A 15 30 min 80V 3A 15 30 min Standard Field Conditions Cooling Recommended PLATE ELECTRODE DISTANCE 4cm 7cm 10cm Buffer Power Run Power Run Power Run conditions Time conditions Time conditions Time Towbin 10V 0 3A 16 Hrs 10V 0 2A 16 Hrs 10V 0 15A 16 Hrs 20V 0 7A 20V 0 4A 20V 0 3A 30V 11 30V 0 7 A 30V 0 5A CAPS 10V 0 35A 4 Hrs 10V 0 2A 4 Hrs 10V 0 2A 4 Hrs 20V 0 8A 20V 0 55 A 20V 0 4A 30V 0 9A 30V 0 6A Carbonate 10V 0 7A 4 Hrs 10V 0 4A 4 Hrs 10V 0 33A 4 Hrs 20V 1A 20V 0 7A 30VAA When running under constant voltage the PowerPac 200 power supply will automatically cross over to constant current if the power supply s current limit is reached 12 Table 3 2 Native Gels These conditions were determined empirically using 12 5 Tris HCl Criterion gels and native horse myoglobin samples See Section 3 3 for buffer formulations High Intensity Field Conditions Cooling Required PLATE ELECTRODE DISTANCE 4cm 7cm 10cm Buffer Power Run P
5. Catalog Number Product description 170 3990 Trans Blot Plus Cell with Plate Electrodes and Super Cooling Coil includes 3 gel holder cassettes cell with lid and power cables 6 fiber pads 1 pack blot absorbent paper 26 5 x 28 cm pack of 30 roller and stir bar 165 5052 PowerPac 200 Power Supply 110 120 V 165 5053 PowerPac 200 Power Supply 220 240 V Trans Blot Plus Cell Accessories 170 3994 Trans Blot Plus Gel Cassette Assembly Tray 170 3995 Fiber Pads 27 x 28 5 cm 2 170 3996 Blot Absorbent Paper 26 5 x 28 cm 60 sheets 162 0251 Nitrocellulose Membrane 0 45um 26 5 x 28cm 10 sheets 162 0252 Nitrocellulose Membrane 0 2um 26 5 x 28cm 10 sheets 162 0253 Supported Nitrocellulose Membrane 0 2um 26 5 x 28cm 10 sheets 162 0254 Supported Nitrocellulose Membrane 0 45um 26 5 x 28cm 10 sheets 162 0255 Immun Blot PVDF membrane 26 5 x 28 cm 10 sheet 162 0256 Sequi Blot PVDF membrane 26 5 x 28 cm 10 sheets 170 3997 Stir bar 170 3998 Trans Blot Plus Roller 6 inch wide 170 3999 Trans Blot Plus Gel Holder Cassette 1 170 4990 Trans Blot Plus Super Cooling Coil 170 4991 Trans Blot Plus Platinum Anode Plate Electrode 170 4992 Trans Blot Plus Stainless Steel Cathode Plate Electrode 170 4995 Trans Blot Plus Cell Buffer Tank 170 4996 Trans Blot Plus Cell Lid with Cables 170 4997 Trans Blot Plus Gel Holder Cassette Clamps 3 29 Section 9 References 1 N On FF WO N Burnette W N Anal Biochem
6. H O to 1 liter Towbin Buffer with 15 Ethanol 25 mM Tris 192 mM glycine 1596 v v ethanol pH 8 3 a Using 10X Tris glycine buffer catalog 161 0734 for 1L bottles or catalog 161 0757 for 5L cube 100 ml of 10X Tris glycine buffer 750 ml of deionized water dd H O 150 ml of ethanol b Using dry reagents 3 03 g Tris 14 4 g glycine 600 ml deionized water dd H O 150 ml of ethanol add dd H O to 1 liter 18 CAPS Buffer CAPS based transfer buffers 10 mM CAPS 10 methanol pH 11 may be preferable for transfers of high molecular weight proteins e g gt 50 000 Da and in cases where the glycine component of Towbin buffer may interfere with downstream protein sequencing applications CAPS Buffer with 20 Methanol 10 mM CAPS 3 cyclohexylamino 1 propane sulfonic acid 20 methanol pH 11 a Using dry reagents 2 21 g CAPS 600 ml deionized water dd H O Adjust to pH 11 with NaOH add dd H O to 800 ml 200 ml methanol CAPS Buffer with 10 Methanol 10 mM CAPS 3 cyclohexylamino 1 propane sulfonic acid 1096 methanol pH 11 Using dry reagents 2 21 g CAPS 600 ml deionized water dd H O Adjust to pH 11 with NaOH add dd H O to 900 ml 100 ml methanol CAPS Buffer with 15 Ethanol 10 mM CAPS 3 cyclohexylamino 1 propane sulfonic acid 15 ethanol pH 11 a Using dry reagents 2 21 g CAPS 600 ml deionized water dd Adjust to pH 11 with NaOH add dd H O to 850 ml 150 ml ethanol 19 Du
7. Table 3 4 RNA These conditions were determined empirically using 5 uniform TBE Criterion gels and the low range Fluorescein labeled DNA standards See Section 3 3 for buffer formulations High Intensity Field Conditions Cooling Required PLATE ELECTRODE DISTANCE 4cm 7cm 10cm Buffer Power Run Power Run Power Run conditions Time conditions Time conditions Time 1X TBE 30V 2A 30 min 44V 2A 45 min 63V 2A 60 min 40V 3A 15 30 min 67V 3A 15 30 min 93V 3A 15 30 min 1X TAE 21V 2A 30 min 35V 2A 45 min 454V 2A 60 min 30V 3A 15 30 min 50V 3A 15 30 min 70V 3A 15 30 min Standard Field Conditions Cooling Recommended PLATE ELECTRODE DISTANCE 4cm 7cm 10cm Buffer Power Run Power Run Power Run conditions Time conditions Time conditions Time 1X TBE 10V 0 6A 16 Hrs 10V 0 35A 16 Hrs 10V 0 25A 16 Hrs 20V 0 85A 20V 0 55A 30V 0 9A 1X TAE 10V 0 8A 16 Hrs 10V 0 5A 16 Hrs 10V 0 35A 16 Hrs 20V 0 8A 3 2 Advice for Electrophoretic Transfer 1 Equilibration of gels All electrophoresis gels should be equilibrated in transfer buffer prior to electrophoretic transfer to remove contaminating electrophoresis buffer salts If salts are not removed they will increase the conductivity of the transfer buffer and the amount of heat generated during the transfer Also gels will shrink or swell to various degrees in the transfer buffer depending on the acrylamide percentage and the buffer compos
8. efficiency of transfer Cooling which is achieved with the Super cooling coil and a refrigerated recirculating water bath is required for high intensity transfers and is recommended for longer overnight transfers The Trans Blot Plus tank is designed to simultaneously accommodate the two plate electrode cards three gel holder cassettes and the Super cooling coil 1 2 Specifications Trans Blot Plus cell tank Overall dimensions Material Buffer requirement Buffer capacity Electrodes Electrode plate dimension Electrode card dimension Material Support card Anode plate Cathode plate Distance anode to cathode Cassettes Cassette dimension Material Blotting area Gel capacity per cassette Gel cassette assembly tray Material Overall dimensions 39 4 cm x 17 27 cm x 30 cm Acrylic 12 liters 14 liters 22 86 cm x 24 45 cm 36 2 cm x 28 26 cm Molded Polycarbonate Platinum coated titanium Stainless steel Adjustable to 4 cm 7 cm or 10 cm 28 x 30 7 cm Molded Polyphthalamide PPA 26 5 x 28 cm 1 PROTEAN Plus PROTEAN II XL or PROTEAN II xi gel 4 Criterion gels or 9 ReadyGel gels Molded PETG 42x42x6 3cm 1 3 Safety Power to the Trans Blot Plus cell is supplied by an external DC voltage power supply This power supply must be ground isolated in such a way that the DC voltage output floats with respect to ground All of Bio Rad s power supplies meet this important safety requirement Regardless of
9. the pH of the following buffers Use only analytical grade methanol because metallic contaminants in low grade methanol will plate onto the electrodes Always add methanol or ethanol last to prevent precipitation Note Some pH meter electrodes will not provide correct measurements of the pH of Tris buffers If the pH of the buffer is incorrect check that the pH meter electrode is designed to work with Tris buffers If the pH meter electrode functions properly for Tris buffers and the pH is below 8 0 remake the buffer 1 Buffers for SDS PAGE gels Towbin Buffer This is a general purpose transfer buffer that was first described by Towbin Towbin Buffer with 20 Methanol 25 mM Tris 192 mM glycine 20 v v methanol pH 8 3 a Using 10X Tris glycine buffer catalog 161 0734 for 1L bottles or catalog 161 0757 for 5L cube 100 ml of 10X Tris glycine buffer 700 ml of deionized water dd H O 200 ml of methanol b Using dry reagents 3 03 g Tris 14 4 g glycine 600 ml deionized water dd H O 200 ml of methanol add dd H O to 1 liter 17 Towbin Buffer with 10 Methanol 25 mM Tris 192 mM glycine 10 v v methanol pH 8 3 a Using 10X Tris glycine buffer catalog 161 0734 for 1L bottles or catalog 161 0757 for 5L cube 100 ml of 10X Tris glycine buffer 800 ml of deionized water dd H O 100 ml of methanol b Using dry reagents 3 03 g Tris 14 4 g glycine 600 ml deionized water dd H O 100 ml of methanol add dd
10. the tank After transfer remove at least half the buffer in the tank before moving the tank off of the magnetic stir plate for cleaning To drain the tank use the drain port fittings provided and an additional length of tubing see Additional Components First be sure that the open end of the tubing is placed into a receptacle that is large enough to accommodate all the buffer Then insert the male quick connect fitting onto the drain port on the side of the tank see Figure 5 The tank will begin to drain as soon as the connection is made Fig 5 2 7 Running Acidic Transfers When transferring under acidic conditions switch the orientation of the gel and membrane or simply reverse the orientation of the cassette so that the red side faces the cathode electrode card black Do not reverse the electrode plates or plug the cables into the reverse poles This will cause irreversible damage to the plate electrodes 10 Section 3 Transfer Conditions 3 1 General Guidelines and Running Conditions The electric field strength V cm is the driving force in electrophoretic transfer Therefore the most efficient transfers are obtained when the distance between the electrodes of a blotting cell is reduced The Trans Blot Plus cell offers three different electrode placements resulting in anode to cathode distances of 4 7 and 10 cm for transfer of one two or three gel holder cassettes respectively In any of these configurations tran
11. 9 65 Fx 01 47 41 9133 Germany Ph 089 318 84 177 Fx 089 318 84 123 Hong Kong Ph 852 2789 3300 Fx 852 2789 1257 India Ph 91 124 6398112 113 114 6450092 93 Fx 91 124 6398115 6450095 Israel Ph 03 951 4127 Fx 03 951 4129 Italy Ph 39 02 216091 Fx 39 02 21609399 Japan Ph 03 5811 6270 Fx 03 5811 6272 Korea Ph 82 2 3473 4460 Fx 82 2 3472 7003 Latin America Ph 305 894 5950 Fx 305 894 5960 Mexico Ph 52 5 534 2552 to 54 Fx 52 5 524 5971 The Netherlands Ph 0318 540666 Fx 0318 542216 New Zealand Ph 64 9 4152280 Fx 64 9 443 3097 Norway Ph 47 23 38 41 30 Fx 47 23 38 41 39 Poland Ph 48 22 8126 672 Fx 48 22 8126 682 Portugal Ph 351 21 472 7700 Fx 351 21 472 7777 Russia Ph 7 095 721 1404 Fx 7 095 721 1412 Singapore Ph 65 2729877 Fx 65 2734835 South Africa 00 27 11 4428508 Fx 00 27 11 4428525 Spain Ph 34 91 590 5200 Fx 34 91 590 5211 Sweden Ph 46 0 8 55 51 27 00 Fx 46 0 8 55 51 27 80 Switzerland Ph 061 717 9555 Fx 061 717 9550 United Kingdom Ph 0800 181134 Fx 01442 259118 Sig 0402 4006224 Rev B
12. Trans Blot Plus Electrophoretic Transfer Cell Instruction Manual Catalog Number 170 3990 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 Table of Contents Page Section 1 General Information censere 1 2IntFOCIUCTIOY aucem e oA P nre 1 1 2 Specifications oe ede eee ee te Let eats 2 195 Safety zocs e ub an ora tete emis iein 3 Section 2 Set Up and Basic Operation of the Trans Blot Plus Cell 5 274 CombpoHenhtss noit hg up ORE tate a a etae aec RE 5 2 2 Additional Components sse nnne 6 2 3 Setting up the Tank and Cooling System 6 2 4 Assembling the Gel Sandwich and 8 2 5 J ranster eiie uo e OUR e I ME fea 9 2 6 Draining the Tank teretes eere eere ete ede erbe 10 2 7 Running Acidic Transfers sessssseeeeeneeeennen nens 10 Section 3 Transfer Conditions cerent 11 3 4 General Guidelines and Running Conditions 11 3 2 Notes on Electrophoretic Transfer 5 15 X3 B fferFormulations ionic iare bera ies 17 Section 4 Strategies for Optimizing Electrophoretic Transfer 21 4 1 Optimizing Protein Transfer
13. acing the Trans Blot Plus cell in the cold room is not an adequate means of controlling transfer buffer temperature The tank of the Trans Blot Plus cell is an effective thermal insulator thus it limits the efficient dissipation of heat 6 Using a stir bar during transfer For all blotting applications a stir bar must be placed inside the Trans Blot Plus cell so that the transfer buffer is stirred during the course of the experiment This will help to maintain uniform conductivity and temperature during electrophoretic transfer Failure to properly control transfer buffer temperature results in poor transfer of macromolecules and poses a potential safety hazard 7 Transfer buffer pH Do not adjust the pH of transfer buffers unless this is specifically indicated Adjusting the pH of transfer buffers when not indicated will result in increased buffer conductivity manifested by higher initial current output and decreased resistance 8 Transfer buffer recommendations Use only high quality analytical grade methanol Contaminated methanol can cause increased transfer buffer conductivity and poor transfer Reusing the transfer buffer is not advised since the buffer will likely lose its ability to maintain a stable pH during transfer Diluting transfer buffers below their recommended levels is also not advised since this will decrease their buffering capacity 9 Voltage limits Do not increase the voltage settings beyond those indicated in Tabl
14. d be considered for optimizing elution in such transfers 1 Alter the gel composition a Lower total monomer or crosslinker for polyacrylamide gels b Lower agarose This allows better elution of high molecular weight DNA 2 Alter the DNA denaturants It has been found that glyoxal denaturation allows more efficient elution of DNA than NaOH Boiling polyacrylamide gels to denature DNA has also been found to give excellent results Base denaturation often causes polyacrylamide gels to weaken and stick to blotting membranes Section 5 Choice of Blotting Membranes 5 1 Protein Blotting Membranes A variety of blotting membranes are available each with particular advantages depending on the needs of the experiment The physical properties and performance characteristics of a membrane should be evaluated in selecting the appropriate transfer conditions Table 5 1 Guide to Protein Blotting Membranes Membrane Pore Size Binding Notes Capacity g cm Nitrocellulose 0 45 um 80 100 General purpose protein blotting 0 2 um membrane Supported 0 45 um 80 100 Pure nitrocellulose cast on an Nitrocellulose 0 2 um inert synthetic support increased strength for easier handling and for reprobing PVDF 0 2 um 170 200 High mechanical strength and chemical stability used for protein sequencing and western blotting low background to signal ration enhanced binding in the presence of SDS Must be wet in alcohol before equilibra
15. e Cards The Trans Blot Plus cell is supplied with a pair of plate electrodes a platinum coated titanium anode and a stainless steel cathode Both electrode cards are removable The anode plate red must be placed into the slot that is nearest the front face of the buffer tank The placement of the cathode plate black is variable the cathode plate may be positioned in either the third fourth or fifth slot 4 cm 7 cm or 10 cm away from the anode for electrophoretic transfers of one two and three gel containing cassettes respectively The electrode cards are held in place in the tank with the nylon screws provided Roller The roller is used to ensure proper contact between gel and membrane and to remove trapped bubbles during sandwich assembly Stir bar A 3 x 7 stir bar is included with the Trans Blot Plus cell and should be used during every electrophoretic run to maintain uniform conductivity and temperature during transfer Drain Port and Quick Connect Fitting The drain port and quick connect fitting on the side of the Trans Blot Plus cell facilitate draining the buffer from the tank after transfer Remove at least half of the transfer buffer volume prior to moving the tank 2 2 Additional Components Magnetic stir plate A magnetic stir plate with a surface area that is sufficient to accommodate the Trans Blot Plus cell is required for constant stirring of transfer buffer during electrophoresis Recommended stir plates incl
16. es 3 1 3 4 for overnight operation Buffer conductivity must be close to the current listed and a current limit should be set on the power supply If overnight transfers at low voltages are ineffective and higher voltages are necessary then decrease the transfer time and use active cooling with the higher voltage settings Failure to decrease transfer time and use cooling may result in a safety hazard 16 10 These variables will change total resistance and current readings Alterations to the buffer make up e g addition of SDS or changes in ion concentration due to the addition of acid or base to adjust the pH of the buffers e Gel pH ionic strength and percentage of acrylamide especially if the gel has not been fully equilibrated e Volume of buffer current increases when volume increases Transfer temperature current increases when temperature increases e Time during the transfer at which reading was taken current normally increases as the buffering capacity diminishes with progress of the run 3 3 Buffer Formulations All recipes in this section make 1 liter of buffer A total of 15 liters is sufficient for transfer and gel cassette assembly for the Trans Blot Plus cell Scale up the following recipes appropriately Note that some buffers can be made as concentrated stock solutions and diluted prior to use Some buffers may also be purchased as pre made concentrated stock solutions Do not add acid or base to adjust
17. ic acid 100 ml 0 5 M EDTA pH 8 0 Add 20 ml 50X stock to 980 ml deionized water dd H O to make a 1X working solution Section 4 Strategies for Optimizing ElectrophoreticTransfer 4 1 Optimizing Protein Transfer Generally quantitative elution of denatured high molecular weight proteins is difficult The following tactics alone or in combination will increase transfer efficiency 1 Improve gel membrane contact Failure of molecules to bind efficiently to the membrane caused by poor gel membrane contact is often confused with inefficient elution of proteins from the gel Poor contact is usually due to excess moisture in the gel membrane interface Use the roller to assure good contact between the gel and membrane Proper selection of filter paper thickness will also help assure good compression Equilibrate the gel and membrane in transfer buffer for at least 15 minutes prior to transfer to prevent shrinking of either component during transfer and to eliminate reactants such as urea or SDS from the gel Increase transfer time An initial control should be performed to determine the time required for complete transfer Times may vary from as little as 15 30 minutes to as long as overnight Remember all overnight applications should be performed at 10 30 Volts to minimize heating problems Increase the power Initial controls should be performed to evaluate the efficiency of increasing the V cm as well as its effects on
18. ition Equilibration allows the gel to adjust to its final size prior to electrophoretic transfer Equilibration is not necessary in situations where the same buffer is used for both electrophoresis and transfer e g native gel transfers 15 2 Current limits The PowerPac 200 Power Supply is capable of a 200 Volt output Unless a current limit is set uncontrolled conductivity changes may result in full power being delivered to the Trans Blot Plus cell The transfer buffer may heat up further increasing conductivity resulting in a potential safety hazard Refer to the PowerPac 200 Power Supply Instruction Manual for setting current limits and run times 3 Polarity of transfer Do not reverse the polarity of the plate electrodes This will result in corrosion and rusting of the stainless steel cathode If this should occur the stainless steel should be cleaned with a mild non abrasive cleanser to remove the rust 4 Dissipating Heat Electrophoretic transfer entails large power loads and consequently heat generation The use of the Super cooling coil and a refrigerated circulating bath is required for high intensity field transfers and is recommended for long unsupervised runs Pre chilling the buffer or the use of ice blocks are common practices for heat dissipation in blotting yet their application for the Trans Blot Plus cell should be limited to only runs lasting less than 1 hr and requiring less than 150 Watts total power Pl
19. late Clamp Fiber Pad Hinge Gel Holder Cassette Black Plate Fig 3 1 For each gel cut one piece of membrane and two pieces of filter paper to the dimensions of the gel Pre cut membranes and filter papers are available see Product Information Equilibrate gels and membranes by soaking them in transfer buffer for 15 minutes Pour 3 liters of transfer buffer into a tray for assembly of the cassettes Place the black cassette plate into the tray with the clamps in their fully extended position 5 Place a fiber pad on the black cassette plate making sure it is thoroughly wet 6 Place a piece of filter paper on top of the fiber pad Make sure there is enough buffer to thoroughly wet the filter paper 7 Carefully place the equilibrated gel on top of the filter paper Note Extra care is required when handling large gels first align one side of the gel with the side of the filter paper and slowly lower the rest of the gel 8 Carefully place the pre soaked membrane on top of the gel Make sure the membrane is properly positioned as it touches the gel To avoid ghost prints or artifacts do not move the membrane after it is positioned Use the roller to remove any air bubbles and to ensure proper contact between the gel and membrane 9 Wetasecond piece of filter paper in transfer buffer and place it on top of the membrane 10 Soak a fiber pad in transfer buffer and place it on top of the filter paper
20. ly by immersion of the sheet in transfer buffer heat distilled water until just under the boiling point and soak the membrane until it is completely wet Equilibrate in transfer buffer until ready for use Poor Binding to the Membrane PVDF 1 The membrane may not be completely wet Because of the hydrophobic nature of PVDF the membrane must be completely soaked in methanol prior to equilibration in aqueous transfer buffer Follow the directions in the product insert 2 The membrane may have been allowed to dry during handling e Acompletely wet membrane has a gray translucent appearance White spots will form on the surface of the membrane indicating that it has been allowed to dry Since proteins will not bind to the dry spots rewet the membrane with methanol and re equilibrate in transfer buffer 3 Proteins may be transferring through the membrane Decrease the voltage if transferring under high intensity conditions Place an additional piece of PVDF membrane in the gel sandwich and analyze this added piece for evidence of proteins that may have transferred completely through the first piece 4 SDSinthe transfer buffer will reduce binding efficiency of proteins Reduce or eliminate the SDS from the transfer buffer Power conditions are too high e Always check the current at the beginning of the run The current may be too high for a particular voltage setting If the buffer is prepared improperly the conduc
21. manual e Stain the gel after transfer with a total protein stain like Coomassie Blue or SYPRO Ruby to make sure that proteins have left the gel Transfer time is too short Increase the transfer time Power is too low e Always check the current at the beginning of the run The current may be too low for a particular voltage setting If the buffer is prepared improperly the conductivity may be too low and not enough power will be delivered to the cell See the power guidelines for specific applications in Section 3 Prepare new buffer or increase the voltage e Try the high intensity blotting option Charge to mass ratio is incorrect native transfers e Tryamore basic or acidic transfer buffer to increase protein mobility Proteins near their isoelectric point will transfer poorly Buffer pH should be 2 pH units higher or lower than the pl of the protein of interest for optimal transfer efficiency Power supply circuit is inoperative or an inappropriate power supply was used e Check the fuse Be sure the voltage and current output of the power supply match the needs of the blotting instrument Methanol in the transfer buffer is restricting elution Reduction of methanol results in increased transfer efficiency of proteins from the gel but it also diminishes binding to nitrocellulose membranes Protein is precipitating in the gel 1 Use SDS in the transfer buffer SDS can increase transfer efficiency b
22. nn carbonate buffer In some cases this buffer may produce higher efficiency transfers and improve the ability of antibodies to recognize and bind to proteins 10 mM NaHCO 3 mM Na CO 20 methanol pH 9 9 a Using dry reagents 0 84 g NaHCO 0 318 g Na CO anhydrous 500 ml deionized water dd H O 200 ml methanol add dd H O to liter 2 Buffers for native gels Towbin Buffer 25 mM Tris 192 mM glycine pH 8 3 a Using 10X Tris glycine buffer catalog 161 0734 for 1L bottles or catalog 161 0757 for 5L cube 100 ml of 10X Tris glycine buffer 900 ml of deionized water dd H O b Using dry reagents 3 03 g Tris 14 4 g glycine 600 ml deionized water dd H O add dd H O to 1 liter 3 Buffers for nucleic acid transfers TBE Tris Borate EDTA 89 mM Tris borate 2 mM EDTA pH 8 3 a Using 10X TBE buffer catalog 4161 0733 for 1L bottles or catalog 161 0770 for 5L cube 100 ml of 10X TBE buffer 900 ml of deionized water dd H O b Using dry reagents 10X stock 108 g Tris base 55 g boric acid 40 ml 0 5 M EDTA pH 8 0 Add 100 ml of the 10X stock to 900 ml deionized water dd H O to make a 1X working solution 20 Tris Acetate EDTA 40 mM Tris Acetate 1 mM EDTA a Using 50X TAE buffer catalog 161 0743 for 1L bottles or catalog 161 0773 for 5L cube 20 ml of 50X TAE buffer 980 ml of deionized water dd H O b Using dry reagents 50X stock 242 g Tris base 57 1 ml glacial acet
23. ond the indicated buffer level lines marked on the tank 7 Place the stir bar in the tank 8 Start cooling Cooling is required for high intensity field conditions and is recommended for prolonged unsupervised runs Place the Super cooling coil into its designated slot at the rear face of the tank Connect the Super cooling coil to the refrigerated circulator according to the manufacturer s instructions Avoid restrictive fittings internal diameter reductions and excessive extensions of tubing Turn on power to the stir plate Note To test that the cooling system is functioning properly before the first transfer run is initiated place the Trans Blot Plus cell on the magnetic stirrer and pour in 11 L of buffer or water With no power applied to the tank the buffer should cool by 5 in approximately 20 min 2 4 Assembling the Gel Sandwich and Cassette Assembly Each gel sandwich will contain the gel and membrane sandwiched between two pieces of blot absorbent filter paper To prevent contamination always wear gloves when handling the gels membranes and filter paper to prevent contamination An optional gel cassette assembly tray is available for the Trans Blot Plus cell see Product Information This tray is large enough to accommodate the gel holder cassette during sandwich assembly The lid of the tray can be used for soaking membranes Fiber Pad Filter Paper Membrane Gel Filter Paper Gel Holder Cassette Red P
24. onsult your electrophoresis manual for more details Gel cassette pattern transferred to blot 1 Contaminated or thin fiber pads are used Replace the fiber pads or thoroughly clean the contaminated pads 2 The transfer buffer is contaminated Make fresh solutions Poor binding to the membrane Nitrocellulose 1 20 methanol in the transfer buffer is generally optimal for protein binding e sure the buffer contains the proper amount of methanol Proteins may be transferring through the nitrocellulose Use PVDF or 0 2um nitrocellulose smaller pore size Decrease the voltage if using the high intensity option Place an additional piece of nitrocellulose membrane in the gel sandwich and analyze this added piece for evidence of proteins that may have transferred completely through the first piece Proteins 15 000 Da may show diminished binding to 0 45 nitrocellulose or may be washed from the membrane during assays Use PVDF or nylon membrane which have higher binding capacities 26 Use Tween 20 detergent in the wash and antibody incubation steps Reduce or eliminate the more stringent washing conditions 4 SDSinthe transfer buffer will reduce binding efficiency of proteins Reduce or eliminate the SDS from the transfer buffer 5 The membrane may not be completely wet e White spots on the membrane indicate dry areas where protein will not bind If wetting does not occur immediate
25. or an authorized agent Use of spare parts supplied by anyone other than Bio Rad Laboratories Damage caused by deliberate or accidental misuse Corrosion due to use of improper solvent or sample oa Use with chlorinated hydrocarbons e g chloroform aromatic hydrocarbons e g toluene benzene or acetone For any inquiry or request for repair service contact Bio Rad Laboratories after confirming the model and serial number of your instrument Warranty Information Model Serial Number Date of Delivery Warranty Period EN61010 is an internationally accepted electrical safety standard for laboratory instruments When running under constant voltage the PowerPac 200 power supply may automatically cross over to constant current if the power supply s current limit has been reached 31 Life Science Group Bio Rad Laboratories Inc Web site www bio rad com Bio Rad Laboratories Main Office 2000 Alfred Nobel Drive Hercules CA 94547 Ph 510 741 1000 Fx 510 741 5800 Also in Australia Ph 02 9914 2800 Fx 02 9914 2889 Austria Ph 01 877 89 01 Fx 01 876 56 29 Belgium Ph 09 385 55 11 Fx 09 385 65 54 Brazil Ph 55 21 507 6191 Canada Ph 905 712 2771 Fx 905 712 2990 China Ph 86 21 63052255 Fx 86 21 53964775 Czech Republic Ph 420 2 4141 0532 Fx 420 2 4143 1642 Denmark Ph 45 44 52 1000 Fx 45 4452 1001 Finland Ph 358 0 9 804 2200 Fx 358 0 9 804 1100 France Ph 01 47 95 6
26. ower Run Power Run conditions Time j conditions Time conditions Time Towbin 50V 2A 45 min 90V 2A 45 min 100V 1 7A 60min no methanol 80V 3A 15 30 100V 2 2A 15 30 min 130V 2 3A 15 30 min Standard Field Conditions Cooling Recommended PLATE ELECTRODE DISTANCE 4 cm 7 cm 10 cm Buffer Power Run Power Run Power Run conditions Time conditions Time conditions Time Towbin 10V 0 3A 16 Hrs 10V 0 2A 16 Hrs 10V 0 15A 16Hrs methanol 20V 0 7A 20V 0 4A 20V 0 3A 30V 1 1A 30V 0 7A 30V 0 5A 13 Table 3 3 Isoelectric Focusing Native Acid Urea Gels Basic Proteins These conditions were determined empirically using 12 5 Tris HCl Criterion gels and native horse myoglobin samples Read about acidic transfers in Section 2 7 of this manual High Intensity Field Conditions Cooling Required PLATE ELECTRODE DISTANCE 4cm 7cm 10cm Buffer Power Run Power Run Power Run conditions Time conditions Time conditions Time 0 7 Acetic 50V 2A 45 min 70V 2A 60 min 100V 1 9A min acid pH 2 8 70V 3A 15 30 min 110V 3A 15 30 min 125V 2 4A 15 30 min Standard Field Conditions Cooling Recommended PLATE ELECTRODE DISTANCE 4cm 7cm 10cm Buffer Power Run Power Run Power Run conditions Time conditions Time conditions Time 0 7 Acetic 10V 0 4A 16 Hrs 10V 0 25A 16 Hrs 10V 0 15A 16 Hrs acid pH 2 8 20V 0 8A 20V 0 55A 20V 0 35A 30V 0 8A 30V 0 55A 14
27. rode card in place with the white nylon screw 3 Position the cathode black electrode card into the third fourth or fifth slot from the front 4 cm 7 cm or 10 cm away from the anode card depending on whether you will be transferring one two or three gel containing cassettes respectively see Figure 2 Tighten the electrode card in place with the white nylon screw Super Cooling Coil Cathode Plate Black Gel Holder Cassette 1 Anode Plate Red Cathode Plate Black Super Cooling Coil One Gel Holder Cassette Loaded Super Cooling Coil pu z j Cathode Plate Black d x i Gel Holder Cassettes 2 Lo V Anode Plate Red 74 b ye Two Gel Holder Cassettes Loaded s Super Cooling Coil c Cathode Plate Black Gel Holder Cassettes 2 Anode Plate Red Gel Holder Cassettes 3 Anode Plate Red Three Gel Holder Cassettes Loaded Fig 2 4 If necessary adjust the position of the cathode leads black on the lid so that they correspond to the position of the cathode electrode plate black within the tank 5 Position the Trans Blot Plus cell on a magnetic stir plate 6 Add transfer buffer to the appropriate fill line Choose the fill line that is appropriate for the number of gel holder cassettes that you are using Note In order to avoid overflow and electrical hazards do not fill the tank bey
28. rts Lid o E o 2 3 o Cathode Plate Black Gel Holder Cassettes 3 Anode Plate Red Buffer Tank Handles Drain Port Fig 1 Section 2 Set up and Basic operation 2 1 Components Buffer Tank and Lid The buffer tank and lid combine to fully enclose the inner chamber during electrophoresis The lid cannot be removed without disrupting the electrical circuit Handles on both sides of the tank facilitate transport Guide marks on the front and back of the tank identify appropriate fill levels for transfer buffer On the inside the tank has 5 separate slots for variable placement of the electrode cards and gel holder cassettes and a designated space for the cooling coil Multiple ports on the lid allow three different connection points for the cathode black electrode card Gel Holder Cassettes Each gel holder cassette has an overall effective blotting area of 26 5 x 28 cm The gel membrane sandwich is placed into the cassette between two fiber pads which are also included The gel holder cassette design includes three separate clamps that ensure even pressure across the gel and membrane sandwich A detachable hinge mechanism prevents gel sandwiches from slipping during assembly Super Cooling Coil Coolant from a refrigerated circulator see Additional Components passes through the Super cooling coil to cool the transfer buffer during high intensity or prolonged runs Electrod
29. sfers may be performed under either high intensity or standard field conditions High intensity field transfers require less than 5 hours to complete Standard field transfers require up to 16 hours to complete and are generally run overnight In order to produce such rapid transfers high intensity transfers require higher power input and consequently produce more heat Use of the Super cooling coil is required for high intensity transfers and is recommended for standard field conditions where the run time is prolonged and usually unsupervised The following are recommended running conditions for a variety of transfer buffers and electrode distances Transfers may be performed under either constant voltage or constant current settings Constant voltage settings provide constant field strength and tend to provide the most efficient transfer Use of the Super cooling coil should prevent heating when transferring under constant voltage Please note that the run times will need to be increased for gradient gels and may need to be decreased if your proteins have a low molecular weight and transfer quickly Transfer conditions should be optimized for every individual application 11 Table 3 1 SDS PAGE Gels These conditions were determined empirically using 12 5 Tris HCl Criterion gels and prestained SDS PAGE molecular weight standards Catalog 161 0318 See Section 3 3 for buffer formulations High Intensity Field Conditions Cooling Required
30. sing effects on the efficacy of trans fer Alcohol in the transfer buffer removes SDS from protein SDS complexes and increases the affinity between proteins and nitrocellulose membranes Alcohol also causes a reduction in gel pore size restricting transfer of some proteins Alcohol may also cause some proteins to precipitate and transfer inefficiently Proteins bind efficiently to PVDF membrane in the absence of alcohol Therefore elimination of alcohol from the transfer buffer and use of PVDF membrane for SDS protein transfers may constitute a logical strategy for analysis of high molecular weight or difficult to transfer proteins5 Alcohol is not required the transfer buffer when proteins are being transferred from gels not containing SDS 5 Alter membrane type As mentioned in 4e PVDF membrane allows transfer in the absence of alcohol PVDF can increase the binding of low molecular weight proteins that sometimes blow through nitrocellulose when transfers are long enough or intense enough to transfer high molecular weight proteins Use Immun Blot PVDF if the blot will be developed with immunochemicals Use Sequi Blot PVDF is the proteins will be sequenced or analyzed by mass spectrometry 22 4 2 Optimizing DNA and RNA Transfer Altering the gel percentage can solve problems with elution of nucleic acids It may be somewhat more difficult to quantitatively transfer large amounts of DNA used in genomic blots The following tactics shoul
31. the temperature of transfer The temperature increase may change buffer resistance and subsequent power delivered as well as the state of protein denaturation thus affecting transfer efficiency 21 4 Vary buffer type and pH a Reduce the buffer strength Dilution of transfer buffer results in lower current at any given voltage This will allow the use of higher voltages without excessive heating b Maximize the charge to mass ratio Alcohols present in SDS transfer buffer strip SDS from proteins Basic proteins in Tris glycine and methanol buffer at pH 8 3 may assume a state near isoelectric neutrality and thus may transfer poorly Buffers with pH of 9 5 to 10 0 have shown much better elution and binding characteristics for basic proteins such as lysozyme and histones Different buffer types at similar V cm may yield different efficiencies Generally Tris buffers allow more efficient transfer than acetate or phosphate buffers Addition of 0 1 SDS detergent to Tris glycine and methanol buffer has been reported to increase transfer efficiency SDS however increases relative current power and heating Temperatures below 10 C may precipitate the SDS so the starting buffer temperature will be higher SDS may also affect the antigenicity of some proteins SDS will aid in eluting the proteins from the gel but it may reduce the binding efficiency of those proteins to the membrane Alcohol in the transfer buffer has oppo
32. tion in buffer Nylon 0 2 um 170 Recommended for nucleic acids 23 PVDF Membrane Bio Rad offers PVDF Polyvinylidene difluoride membranes that are ideal for immunoassays of blotted proteins Immun Blot PVDF or amino terminal sequencing and amino acid analysis Sequi Blot PVDF PVDF retains proteins under extreme conditions such as exposure to organic solvents or acidic or basic conditions Greater protein binding capacity allows for better retention of easily transferred proteins while allowing more time or higher voltages to transfer difficult or larger proteins Greater protein retention during sequencing manipulations enhances the likelihood of obtaining information from rare low abundance proteins by increased initial coupling and more consistent yields In addition PVDF membrane exhibits better binding efficiency of blotted material in the presence of SDS in the transfer buffer PVDF must first be wetted in 10096 methanol but can then be used in a transfer buffer that does not contain alcohol Nitrocellulose Membrane Nitrocellulose membranes have been used extensively for protein binding and detection 8 10 Nonspecific protein binding sites are easily and rapidly blocked on nitrocellulose avoiding subsequent background problems No pre activation of the membrane is required With nitrocellulose low molecular weight proteins especially those 20 000 Da may be lost during post transfer washes thus limiting detection sensiti
33. tivity may be too high resulting in excessive power delivered to the cell See the power guidelines for specific applications in Section 3 Immune Specific Detection Overall high background low signal or lack of development of positive control Consult instructions for immune detection kit or reagents Total Protein Detection Consult stain or detection kit user manual 27 Section 7 Maintenance Cleaning e After transfer remove at least half the buffer remaining in the tank before attempting to lift or move the tank from the magnetic stir plate for cleaning See Section 2 6 for instructions e Use mild soap and warm water to clean the electrodes cassettes and buffer tank Take special care when cleaning the electrode cards or plate electrodes Do not use abrasives or strong detergents Avoid scratching or marring the platinum plate The cathode plate stainless steel can be cleaned with a mild abrasive to remove salt that may be deposited during normal operation e Rinse fiber pads thoroughly under hot water and then in distilled deionized water Improper cleaning of the fiber pads may lead to the appearance of artifacts on subsequent blots Chemical compatibility Trans Blot Plus cell components are not compatible with chlorinated hydrocarbons e g chloroform aromatic hydrocarbons e g toluene benzene or acetone Use of organic solvents voids all warranties 28 Section 8 Product Information
34. ude the PC 610 from Corning the Cimarec 3 from Thermolyne and the Vel A from Cole Parmer Refrigerated circulator The Trans Blot Plus cell requires a refrigerated circulator to work with the Super cooling coil for optimal results The recommended minimum cooling capacity of the refrigerated circulator is 300W at 20 C and minimum pump flow rate is 4 L min Recommended chillers include the Model RTE 7 from Thermo NESLAB and the Model WKL 26 from Thermo Haake Tubing Tubing with a 3 8 internal diameter is required to connect both ends of the Super cooling coil to the refrigerated circulator and for draining the tank using the quick release fitting and drain port Gel Cassette Assembly Tray The optional gel cassette assembly tray is large enough to accommodate a gel holder cassette and the buffer required for sandwich assembly and may be purchased separately see Product Information 2 3 Setting up the tank and cooling system 1 Prepare the transfer buffer See Section 3 3 for buffer formulations Generally 15 liters of transfer buffer will suffice for electrophoresis gel equilibration and sandwich assembly 2 Position the anode red electrode card into the tank in the slot that is nearest the front face of the tank see Figures 1 and 2 This is the only position possible for the anode plate since it is the only position that will provide a connection between the plate electrode and the anode leads in the lid Tighten the elect
35. ut it can also reduce binding efficiency to nitrocellulose and affect reactivity of some proteins with antibodies Eliminate alcohol from the transfer buffer see Section 4 25 Swirls or missing bands diffuse transfers 1 Poor contact between the membrane and the gel Air bubbles or excess buffer remain between the blot and gel Use the roller carefully to roll over the membrane in both directions until air bubbles or excess buffer is removed from between gel and membrane and complete contact is established Use thicker filter paper in the gel membrane sandwich Replace the fiber pads Pads will compress and degrade with time and will not hold the membrane to the gel The membrane is not properly wet or has dried out e White spots on nitrocellulose membrane indicate dry areas where protein will not bind If wetting does not occur immediately by immersion of the sheet in transfer buffer heat distilled water until just under the boiling point and soak the membrane until completely wet Equilibrate in transfer buffer until ready for use Because of the hydrophobic nature of PVDF the membrane must be prewet in methanol prior to equilibration in aqueous transfer buffer Follow the directions in the product insert The gel electrophoresis may be at fault e Artifacts of electrophoresis may occur as a result of poor gel polymerization inappropriate running conditions contaminated buffers sample overload etc C
36. vity Smaller pore size nitrocellulose membrane 0 2 um has been shown to be effective in eliminating this loss Large proteins those gt 100 000 Da that are denatured by SDS may transfer poorly to nitrocellulose if alcohol is added to the transfer buffer Alcohol in the transfer buffer increases binding of SDS proteins to nitrocellulose but decreases pore sizes in the gel 5 2 DNA and RNA Blotting Membrane Zeta Probe Nylon Membrane Zeta Probe membrane is an ideal alternative to nitrocellulose for the analysis of nucleic acids The membranes bind nucleic acids in low ionic strength buffers making electrophoretic transfer of nucleic acids from agarose and acrylamide gels possible Zeta Probe membrane allows efficient binding of all sizes of single stranded DNA and RNA in the presence of low ionic strength buffers Unlike nitrocellulose Zeta Probe membranes can be hybridized as many as 20 consecutive times 24 Section 6 Troubleshooting Guide Poor transfer of proteins 1 Transfer apparatus is assembled incorrectly and the proteins are moving in the wrong direction e gel membrane sandwich may be assembled in the wrong order or the cassette may be inserted in the tank with the wrong orientation Check the polarity of the connections to the power supply Detection system is not working or is not sensitive enough Include proper positive and negative control antigen lanes to test for kit sensitivity Consult kit
37. which power supply is used the maximum specified operating parameters for the cell are 300 VDC Maximum voltage limit 300 Watts Maximum power limit 40 C Maximum ambient temperature limit Current to the cell provided from the external power supply enters the unit through the lid assembly providing a safety interlock to the user Current to the cell is broken when the lid is removed Do not attempt to circumvent this safety interlock Always turn the power supply off before removing the lid or when working with the cell in any way The Trans Blot Plus is certified to meet EN61010 1 safety standard for safety of laboratory equipment Certified products are safe to use when operated in accordance with the instruction manual This safety certification does not extend to other equipment or accessories not EN61010 1 certified even when connected to the Trans Blot Plus This instrument should not be modified or altered in any way Alteration of this instrument will void the manufacturer s warranty void the EN61010 1 safety certification and create a potential safety hazard for the user Bio Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not performed by Bio Rad or an authorized agent EN61010 1 is an internationally accepted electrical safety standard for laboratory instruments Trans Blot Plus Cell Assembly of Pa
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