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1. Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Note To insure best performance from the Model 111 Mini IEF Cell become fully acquainted with these operating instructions before using the cell to separate samples Bio Rad recommends that you first read these instructions carefully Then assemble and disassemble the cell completely without casting a gel After these preliminary steps you should be ready to cast and run a gel Bio Rad also recommends that all Model 111 Mini IEF Cell components and accessories be cleaned with a suitable laboratory cleaner such as Bio Rad Cleaning Concentrate catalog number 161 0722 and rinsed thoroughly with distilled water before use Model Catalog No Date of Delivery Serial No Invoice No Purchase Order No Warranty Bio Rad Laboratories warrants the Model 111 Mini IEF Cell against defects in materials and workmanship for 1 year If any defects occur in the instrument during this warranty period Bio Rad Laboratories will repair or replace the defective parts free The following defects however are specifically excluded Defects caused by improper operation N2. Dp 16 6 6 Set Up Procedure ee cena ere te CRM 17 6 7 Run 5 eerte EEN ENEE AER AA 17 6 8 Sample Detection nter eth ee UE eee Po E 18 Section 7 Troubleshooting nennen nnne 19 7 1 General Troubleshooting esses enne eene 19 7 2 Casting Troubleshooting Polyacrylamide 20 7 3 Casting Troubleshooting Agarose Gelee 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 8 22222 2 22 40 2220202000 000000000000 22 Section 9 Equipment and Accessories sse 23 9 1 Equipment sice 22 0227 C 22 9 3 Gel Support Film techn eere TEE CE E RE Eee 22 9 4 Isoelectric Focusing Chemicals and Reagentz sse 22 05 Bio Lyte Ampholytes EN 23 9 0 OD MP HM 24 9 7 Sample Preparation eere ce E REENEN Ed 24 9 8 Power Supplies ie hte osteitis aes 24 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 1 General Information 1 1 Introduction Bio Rad s Model 111 Mini IEF Cell introduces a simple and innovative inverted gel format for analy
3. Repair or modification done by anyone other than Bio Rad Laboratories or an authorized agent Use of fittings or other spare parts supplied by anyone other than Bio Rad Laboratories Damage caused by accident or misuse Damage caused by disaster DN 5 Corrosion due to use of improper solvent or sample This warranty does not apply to parts listed below 1 Platinum wire glass plates For any inquiry or request for repair service contact Bio Rad Laboratories after confirming the model and serial number of your instrument Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Table of Contents Section 1 General Information esee enne 1 1 1 Introduction occ cccccesssccecessnseceececssseeseecsesscececesessseeececessaaseeececsaasseceeesensaeees 1 1 2 8 iem UBRO nene 1 1 35 ERE 2 14 Model 111 Mini IEF Cell Components eene 3 1 5 Capillary Thin Layer Gel Casting 3 Section 2 Introduction to Isoelectric Focusing seen 4 2 1 Blectrofocusmg Principle 4 2 2 ere aee er Dee br re te aoi 4 2 3 Choice of Support E sk epo ch 5 Section 3 Polyacrylamide Gel Isoelectric Focusing 0000000000000o000000000000000000e 5 3 1 Considerations in Matr
4. 4 rtisan tisan Technology Group is your source for quality Fra op new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com 7 information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED Contact us 888 88 SOURCE sales artisantg com www artisantg com Model 111 Mini IEF Cell Instruction Manual Catalog Numbers 170 2975 and 170 2976 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723
5. 1000 500 Power Supply measures current to the microampere range This expanded monitoring of the changes in current allows better estimation of the time the current stabilizes and the experiment is finished 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 5 Sample Detection and Gel Storage 5 1 Removing the Gel 1 After electrofocusing is complete turn off the power supply and unplug the power cables 2 Slide the protective lid from the cell and remove the gel from the electrodes At this point separate the gel gel support film from the glass plate 3 Remove the Graphite Electrodes and clean with water and a tissue Rinse the chamber with distilled water Put the electrodes back into the cell to prevent damage during storage Cleaning the electrodes immediately after the run will increase their life span 5 2 Band Detection In general proteins are detected by fixing and staining If the proteins are not fixed immediately the high resolving power of electrofocusing can be lost to diffusion Small proteins and proteins with basic pls are particularly difficult to fix Autoradiography biological activity group specific staining and immunological methods are a few examples of alternative detection techniques currently being used This section gives two recommended staining procedures for electrofocusing using Coomassie blue R 250 which has a detection limit for mos
6. 416 624 3019 China Bio Rad Laboratories Yanshan Hotel Office Tower 1307 138A Haidian Road Beijing e Phone 2563146 e Fax 2564308 France Bio Rad S A 94 96 rue Victor Hugo 220 94203 Ivry Sur Seine Cedex e Phone 01 49 60 68 34 Fax 01 46 71 24 67 Germany Bio Rad Laboratories GmbH HeidemannstraBe 164 Postfach 45 01 33 D 8000 M nchen 45 Phone 089 318 84 0 Fax 089 318 84 100 Italy Bio Rad Laboratories Sr Via Cellini 18 20090 Segrate Milano e Phone 02 21609 1 Fax 02 21609 399 J apan Nippon Bio Rad Laboratories K Sumitomo Seimei Kachidoki Bldg 5 3 6 Kachidoki Chuo Ku Tokyo 104 e Phone 03 3534 7515 Fax 03 3534 8027 The Netherlands Bio Rad Laboratories B V Fokkerstraat 10 3905 KV Veenendaal e Phone 08385 40666 e Fax 08385 42216 New Zealand Bio Rad Laboratories Pty Ltd P Box 100 051 North Shore Mail Centre Auckland 10 Phone 09 443 3099 e Fax 09 443 3097 Pacific Bio Rad Laboratories Unit 1111 11 F New Kowloon Plaza 38 Tai Kok Tsui Road Tai Kok Tsui Kowloon Hong Kong e Phone 7893300 e Fax 7891257 Scandinavia Bio Rad Laboratories Kanalv gen 10C 19461 Upplands V sby Sweden Phone 46 0 8 590 73489 Fax 46 0 8 590 71781 Spain Bio Rad Laboratories S A Avda Valdelaparra 3 Pol Ind Alcobendas E 28100 Alcobendas Madrid e Phone 91 661 70 85 Fax 91 661 96 98 Switzerland Bio Rad Laboratories AG Kanalstrasse 17 8152 Glattbrugg e Phone 01 810 16 77 Fax 01 810
7. Bis N N Methylene bis acrylamide 50 g 161 0501 Riboflavin 5 Phosphate 10 g 161 0700 Ammonium Persulfate 10 g 161 0800 TEMED 5 ml 161 0801 TEMED 50 ml 162 0022 Zero m Agarose 10 g 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 9 5 Bio Lyte Ampholytes Catalog Number Description 163 1112 Bio Lyte 3 10 Ampholyte 40 10 ml 163 1132 Bio Lyte 3 5 Ampholyte 20 10 ml 163 1142 Bio Lyte 4 6 Ampholyte 40 10 ml 163 1152 Bio Lyte 5 7 Ampholyte 40 10 ml 163 1192 Bio Lyte 5 8 Ampholyte 40 10 ml 163 1162 Bio Lyte 6 8 Ampholyte 40 10 ml 163 1172 Bio Lyte 7 9 Ampholyte 40 10 ml 163 1182 Bio Lyte 8 10 Ampholyte 20 10 ml 163 1113 Bio Lyte 3 10 Ampholyte 40 25 ml 163 1143 Bio Lyte 4 6 Ampholyte 40 25 ml 163 1153 Bio Lyte 5 7 Ampholyte 40 25 ml 163 1193 Bio Lyte 5 8 Ampholyte 40 25 ml 163 1163 Bio Lyte 6 8 Ampholyte 40 25 ml 9 6 Stains Catalog Number Description 161 0400 Coomassie Blue R 250 10 g 161 0406 Coomassie Blue G 250 10 g 161 0417 Crocein Scarlet 10 g 161 0443 Silver Stain Kit includes oxidizer concentrate silver reagent concentrate and developer enough to stain approximately 48 mini IEF gels 161 0449 Silver Stain Plus Kit includes fixative enhanced concentrate silver complex solution reduction moderator sloution image development reagent development accelerator reagent enough to stain ap
8. temperature Alternatively the gel can be carefully dried with a heat gun on a low heat setting Dried gels can be stored in plastic photograph holders or taped directly into notebooks gel side down 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 6 Agarose Gel Electrofocusing 6 1 Introduction Agarose isoelectric focusing separates large proteins and antibodies that cannot be readily characterized on polyacrylamide IEF gels due to polyacrylamide s smaller pore sizes Molecules greater than 200 000 daltons can easily be separated on a 146 agarose gel The agarose gels are formed within minutes by simply heating the agarose mixture and pouring it into the casting tray much the same way as casting acrylamide gels Sorbitol and glycerol are incorporated into the agarose gel to increase gel viscosity and to counteract electroendosmosis EEO a major cause of sample smearing EEO is the cathodic flow of water in the neutral and alkaline parts of the gel caused by the low concentration of fixed charge carboxyl groups on the gel matrix These carboxyl groups are not charged at pH 3 5 or lower but acquire their full charges at pH 5 5 and higher As a consequence gel shrinkage occurs at the pK of the carboxyl groups resulting in flooding of water and solutes at the alkaline portion of the gel EEO decreases when gel viscosity increases The procedure for agarose isoelectric
9. where g acrylamide g crosslinker T 100 total solution volume in ml g crosslinker C x 100 g acrylamide g crosslinker This formulation will give a suitable non sieving gel for proteins up to 106 daltons that is still rigid enough to handle conveniently A slightly stronger gel of T 5 C 4 may be used for protein samples under 200 000 daltons The choice of a catalyst is extremely important in electrofocusing since any residual ions will affect the final attainable voltage and can lead to overheating and gross distortions in the gel For this reason a three phase catalyst system of ammonium persulfate riboflavin 5 phosphate and TEMED is recommended This system catalyzed by light will give reproducible polymerization with a minimum of ionic contamination The formation of polyacrylamide gels has been extensively studied and a detailed discussion of practical considerations is available in Bio Rad s bulletin 1156 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 2 Stock Solutions for Polyacrylamide IEF Gels Always use high quality distilled or deionized water to prepare stock solutions for electrofocusing 1 Monomer concentrate 25 3 24 25 w v acrylamide 0 75 w v bis N N Methylene bis acrylamide Dissolve 24 25 g acrylamide and 0 75 g bis in water bring to a final volume of 100 ml an
10. 19 33 United Kingdom Bio Rad Laboratories Ltd Bio Rad House Maylands Avenue Hemel Hempstead Herts HP2 7TD Phone 0800 181134 e Fax 0442 259118 Printed in USA M1702975 Rev B Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 4 rtisan tisan Technology Group is your source for quality Fra op new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com 7 information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED Contact us 888 88 SOURCE sales artisantg com www artisantg com
11. alicylic acid SSA 4 To insure a clear gel background take the agarose gel directly from the fixative solution to a 95 ethanol bath Immerse the gel in ethanol for 30 minutes with occasional swirling 5 After the ethanol wash place the gels on a level surface Soak one piece of filter paper in ethanol and place it on top of the gel Then place additional dry filter paper 8 10 sheets or folded paper towels on top of the ethanol soaked paper Press the gel with a 1 kg weight for 30 minutes 6 After pressing dry the gel completely with an air blow dryer or under a laboratory hood s fan 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 7 Autoradiography biological activity group specific staining and immunological techniques are just a few examples of many detection techniques currently used The typical IEF stain is Coomassie brilliant blue R 250 Stain 0 2 Coomassie brilliant blue R 250 28 isopropanol or ethanol 14 acetic acid Filter the stain before use The gel should be stained a minimum of 30 minutes at room temperature 8 Destaining the gels should take only 30 minutes at room temperature Destain Solution 28 isopropanol or ethanol 14 acetic acid 9 Air dry the gel plate 18 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 7 Troubleshooting 7 1 General Troubleshooting Pr
12. ar separation Bio Lyte ampholytes are specifically blended to produce a linear gradient within the stated range and no further blending is generally needed Particular separations however may require a combination of two or more ampholytes to achieve a desired result 3 4 Use of Gel Support Film for Polyacrylamide 1 Remove a sheet s of Gel Support Film for Polyacrylamide catalog number 170 2983 from the package and then reseal the package Polyacrylamide gel support film is sensitive to light over prolonged periods and must be kept sealed in the package until use After the gel has adhered to the support film the film is no longer light sensitive 2 Polyacrylamide gel support film has two surfaces a treated hydrophilic side which the acrylamide adheres to and a hydrophobic surface The sheets are packed with printed interleaf paper which protects the treated surface All sheets are packed treated side up A drop of water will bead on the hydrophobic surface and spread on the hydrophilic surface 3 Pipet a few drops of water onto the clean glass plate 4 Place the hydrophobic side of the gel support film against the plate 5 Roll the gel support film flat with a test tube or similar object to force out excess water and air bubbles 6 Carefully wipe or blot off any excess liquid at the edges The gel support film is now ready for use and should be used as soon as possible Note Basic ampholytes pH gt 8 0 have been found t
13. as Lubrol is a registered trademark of C I Organics Inc 22 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 9 Equipment and Accessories 9 1 Equipment Catalog Number Description 170 2975 Model 111 Mini IEF Cell includes outer chamber and interlocking lid 2 Graphite Electrodes gel casting tray 5 Glass Plates Gel Support Film for Acrylamide 50 sheets 5 Sample Templates 5 ml of Bio Lyte 3 10 ampholyte and instructions 170 2976 Model 111 Mini IEF Cell same as above without casting tray 9 2 Accessories Catalog Number Description 170 2980 Graphite Electrodes 2 170 2981 Capillary Thin Layer Gel Casting Tray 170 2982 Glass Plates 125 x 65 x 1 5 mm 5 170 2985 Sample Templates 5 170 4220 Photopolymerization Light 110 V 170 4242 Photopolymerization Light 220 V 170 4257 Sample Application Filter Paper 200 pieces 9 3 Gel Support Film Catalog Number Description 170 2983 Gel Support Film for Acrylamide 125 x 65 mm 50 sheets 170 2984 Gel Support Film for Agarose 125 x 65 mm 50 sheets 9 4 Isoelectric Focusing Chemicals and Reagents Catalog Number Description 161 0310 IEF Standards pl 4 6 9 6 161 0100 Acrylamide 99 9 100 g 161 0101 Acrylamide 99 9 500 g 161 0107 Acrylamide 99 9 1 kg 161 0103 Acrylamide 99 9 2 kg 161 0108 Acrylamide 99 9 5 kg 161 0200 Bis N N Methylene bis acrylamide 5 g 161 0201
14. cedure will result in poor resolution One advantage of using Bio Rad s PowerPac 3000 or Model 1000 500 power supply is that either power supply can be programmed to carry out this stepped increase in voltage automatically Typical running conditions for various ranges of Bio Lyte ampholytes in the Model 111 Mini IEF Cell are shown in Table 4 1 Any gross variation from these values could indicate problems associated with polymerization buffer preparation etc Table 4 1 Typical Running Conditions Bio Lyte pH Current Power Current Power Current Power Range at 100 V at 200 V at 450 V 3 10 5 6 mA 0 5 0 6 W 5 6 mA 1W 4 mA 2 W 4 6 3 0 3 W 3 4 0 6 0 8 W 4 5 2 5 7 3 0 3 W 3 4 0 6 0 8 W 4 5 2 6 8 3 0 3 W 3 4 0 6 0 8 W 4 5 2 7 9 3 0 3 W 3 4 0 6 0 8 W 4 5 2 8 10 10 mA 1W 20 mA 4 W 4 mA 2 W 5 good way to monitor the progress of isoelectric focusing experiment is to observe the migration of visible marker proteins of known pl to their isoelectric points The IEF Standards catalog number 161 0310 provide eight natural proteins including four visible proteins that are clearly discernable during an IEF run These markers are useful with all non denaturing IEF buffer systems As the focusing nears completion the current will decrease from the values listed in the 450 V column This is a general indication that the focusing is near completion Note The Model
15. d filter through a 0 45 filter Store protected from light at 4 C This solution may be stored up to 1 month 2 0 1 w v riboflavin 5 phosphate FMN 50 mg riboflavin 5 phosphate 50 ml water This solution may be stored up to month at 4 C protected from light 3 10 w v ammonium persulfate 100 mg ammonium persulfate 1 ml water Prepare fresh daily Make sure that the ammonium persulfate is completely dissolved before using 4 25 glycerol w v Add 25 g glycerol to 50 ml H5O Dilute to 100 ml with H 0 5 TEMED N N tetramethylene ethylenediamine Use TEMED neat from the bottle Use only pure distilled TEMED Store cool dry and protected from light 3 3 Reagents for Polyacrylamide Electrofocusing Gels The following volumes will produce sufficient reagent for two 125 x 65 x 0 4 mm gels Monomer ampholyte solution HO 5 5 ml Monomer concentrate 25 T 3 C 2 0 ml 25 glycerol 2 0 ml Ampholyte 0 5 ml Catalyst solutions 10 w v ammonium persulfate 15 ul 0 1 w v FMN 50 ul TEMED neat 3 ul Volume required for 40 ampholyte solutions Bio Lyte 3 10 4 6 5 7 6 8 7 9 ampholytes For 20 ampholyte Bio Lyte 3 5 8 10 ampholytes add 1 ml ampholyte and reduce 2 volume to 5 0 ml Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Note The selection of appropriate ampholyte is determined by the pH range required for a particul
16. ed out under constant voltage conditions in a stepped fashion Begin focusing at 100 V for 15 minutes 2 Increase voltage to 200 V for 15 minutes 3 Finally increase the voltage to 450 V for an additional 60 minutes Note Step increases of voltage are necessary to prevent overheating and subsequent dehydration of the gel Failure to follow this procedure will result in poor resolution 4 A good way to monitor the progress of an isoelectric focusing experiment is to observe the migration of visible marker proteins of known pl to their isoelectric points The IEF Standards catalog number 161 0310 provide eight natural proteins including four visible proteins that are clearly discernable during an IEF run These markers are useful with all non denaturing IEF buffer systems 5 As the focusing nears completion the current will decrease substantially This is a general indication that the focusing is near completion 6 8 Sample Detection 1 After the focusing is complete turn off the power supply and disconnect the power cables from it 2 Slide the lid off the Model 111 Mini IEF Cell and carefully remove the gel from the Graphite Electrodes Rinse the electrodes with water and wipe them gently with a tissue Replace the electrodes in the cell 3 Place the gel gel support film Glass Plate is removed at this point into fixative solution for 15 minutes Fixative Solution 30 methanol 596 trichloroacetic acid TCA 3 5 sulfos
17. ethanol 7 acetic acid 0 5 5 Dissolve the cupric sulfate in water before adding the alcohol Immerse the gel in two or three 500 ml changes of this solution until the background is nearly clear Gentle agitation and slight heating will speed the destaining process Second destaining solution 25 isopropanol or ethanol 7 acetic acid Immerse the gel in this solution to remove the last traces of stain and 50 Note Prolonged soaking of gels with gel support film backings in acidic solutions may cause the gel to separate from the backing Staining and destaining steps should be no longer than 3 4 hours 5 4 Other Detection Methods 1 Coomassie Blue G 250 Quick Stain This technique is nearly as sensitive as Coomassie blue R 250 staining but requires no destaining and will not stain ampholytes It cannot be used in the presence of detergents except urea 3 596 perchloric acid 0 025 Coomassie blue G 250 Immerse gels in this solution for 1 hour Place in 796 v v acetic acid for intensification and preservation Ultrasensitive Silver Stain Bio Rad s Silver Stain catalog number 161 0443 is 10 to 50 times more sensitive than Coomassie blue see Bulletin 1089 and is compatible with both supported and unsupported gels 5 5 Gel Drying and Preservation Place the destained gel in a dust free area with good ventilation a fume hood is excellent for this purpose and allow the gel to dry overnight at room
18. f the polymerized gel The colored portion of the template should coincide with the longer dimension of the gel The application position for the sample varies see Section 3 8 but allow 1 cm at both the top and bottom of the gel for the gel to contact the electrodes Apply samples using a pipettor capable of delivering between 0 5 ul and 2 ul Volumes above 2 ul are not generally recommended for use with the sample template Allow samples to diffuse into the gel for 5 minutes Carefully remove the template from the gel Note For larger samples one may custom form application strips from filter paper or use Bio Rad s Sample Application Pieces catalog number 170 4257 Apply the sample to the filter paper and then place the filter paper at the point of application on the gel Allow the sample to diffuse into the gel for 5 minutes The disadvantage of this method is that some proteins may not be completely eluted from the strip to the gel 3 8 Position of Application There are no fixed rules regarding the positioning of the sample on the gel Samples should not in general be applied to areas where protein bands are expected to focus To protect the proteins from extreme pH exposure the samples should not be applied closer than 1 cm from either electrode The best strategy for a new protein is to make three points of application one at each end and one near the middle of the gel and observe the resulting focusing pattern Section 4 Runn
19. focusing is simple consisting of the following seven steps Each of these steps is described in detail in Sections 6 3 through 6 6 1 Cast the agarose gel Dehydrate the gel to remove excess liquid Focus the gel for 90 minutes Immerse the gel in fixative solution Immerse the gel in ethanol to remove background Immerse the gel in stain Sl Oy cem ums e Destain the gel 6 2 Preparing Agarose Gels Agarose IEF gel contains 1 agarose zero M 2 ampholytes 596 sorbitol 10 glycerol 1 Add 0 5 grams of Bio Rad s Zero M Agarose and 2 5 grams of sorbitol to 20 ml of 25 glycerol and 10 ml of distilled water Place a stir bar into the flask and immerse the flask in a beaker of water Heat the water to boiling 100 C and stir to dissolve the components 30 minutes 2 Place the casting tray into a prewarmed 55 C oven and allow it to equilibrate The tray should remain in the oven until just prior to pouring the gels 3 Turn off heat and add ampholytes while stirring the agarose mixture 2 5 ml of 4096 ampholytes or 5 ml of 20 ampholytes Add more hot 100 C distilled water for a final volume of 50 ml 4 Allow solution to cool to about 55 C before pouring agarose A hot water bath set to 55 C is optimal 14 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Note Do not heat the casting tray to more than 55 C Extended heating above this temperatu
20. hould not in general be applied in areas where protein bands are expected to focus To protect the proteins from extreme pH exposure the samples should not be applied closer than 1 cm from either electrode The best strategy for a new protein is to make three points of application one at each end and one near the middle of the gel and observe the resulting focusing pattern 6 6 Set Up Procedure 1 Slide the lid of the Model 111 Mini IEF Cell toward the electrode plugs to remove it 2 Remove the Graphite Electrodes from the cell and rinse them with distilled water to remove traces of agarose from the previous run The electrodes may be gently wiped with a laboratory tissue if necessary Place the electrodes back into the cell 16 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 Lightly moisten the Graphite Electrodes with water Turn the gel with the adsorbed samples upside down and place it directly on top of the electrodes Position the gel carefully the first time as re positioning may damage the gel surface Do not remove the glass plate from the gel gel support backing because its weight insures good contact between the gel and the electrodes 4 Carefully slide the lid back onto the Model 111 Mini IEF Cell Plug the power cab cell into a power supply able to generate 500 V constant voltage The cell is now ready to separate samples 6 7 Run Conditions 1 Focusing is carri
21. imary amine ampholytes especially basic species contribute to short chain polymer formation like the polymerization seen when high levels of TEMED are used Reduce ampholyte concentration to 1 do not degas or add polymerization inhibitor Only use TEMED when combined with APS and riboflavin 5 PO4 20 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Cause Solution b Polymerization time b Optimize polymerization time is too long causing by placing excess solution in gel to dry onto plastic a 12 x 75 mm glass test tube at the edges of the plate and putting the test tube under Especially true with gels the polymerization light polymerized with APS Remove plate 15 minutes after test tube solution gels c Incorrect procedure used c Do not slide plate sideways to lift plate from casting before removing it Do not tray pry the plate up with fingers Twist the spatula to raise the plate edge nearest the spacer This allows air to penetrate between the gel and the plastic without forming a vacuum d Dirty glass plates and d Wash plates and casting tray casting tray with Bio Rad s Cleaning Concentrate and rinse them thoroughly with deionized or distilled water e Use of wrong side of gel e Gels must be cast on the support film treated hydrophilic side of gel support film f Use of Bio Lyte 8 10 f Adhesion is decreased when ampholyte us
22. ing ampholytes with a pH 8 0 Increase ammonium persulfate to 0 7 mg ml 7 ul of 10 APS stock per ml solution and TEMED at 1 ul ml g Gel support film exposed g Always keep gel support film to light for extended sealed in its light proof periods package Store at room temperature 7 3 Casting Troubleshooting Agarose Gels Problem Cause Solution 1 Gel does not adhere to a Use of incorrect side of a Always cast gels on treated gel support film sticks to gel support film hydrophilic surface of gel casting tray support film b Dirty glass plates and b Wash plates and casting tray casting tray with Bio Rad s Cleaning Concentrate then rinse thoroughly with deionized or distilled water c Casting tray not c Allow 15 30 minutes for equilibrated to 55 C casting tray to equilibrate to 55 C in oven d Agarose solution too hot d For maximum adhesion cast or too cold 21 agarose solutions at 55 C Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 8 References 1 Righetti P G Isoelectric Focusing Theory Methodology and Applications Elsevier Biomedical Press Amsterdam 1983 2 Righetti and Drysdale J W Isoelectric Focusing North Holland Publishing Company Amsterdam 1976 3 Radola B J Modern Methods in Protein Chemistry 21 Walter de Gruyter amp Co New York 1983 Triton is a registered trademark of Rohm and Ha
23. ing process must be stabilized against convection and to a lesser extent diffusion by a support matrix This can be anything from a liquid column stabilized by a sucrose density gradient to a gel cast from agarose or polyacrylamide The principal criteria for a good support matrix are that it should be relatively non sieving so that molecular size is not a factor in protein mobility and that it must be free of charged groups which would give rise to internal fluid flow and distortion of the pH gradient A complete discussion of electrofocusing matrices is given in Reference 1 For analytical work both agarose and polyacrylamide gels provide good supports for electrofocusing Agarose has the advantage of very large pore structures as large as 500 nm making it an ideal non sieving medium however it suffers from varying degrees of residual negative charge from sulfate groups For this reason only agarose proven for electrofocusing applications should be used Bio Rad s Zero M Agarose catalog number 162 0022 Agarose concentrations may vary between 0 5 and 1 25 Proteins as large as 50 x 106 daltons have been successfully electrofocused in agarose Section 3 Polyacrylamide Gel Isoelectric Focusing 3 1 Considerations in Matrix Preparation Because they are prepared from monomers polyacrylamide gels can be tailored to meet particular separations requirements The most common gel composition for horizontal electrofocusing is T 5 C 3
24. ing the Gel 4 1 Set Up Procedure 1 2 Slide the lid of the Model 111 Mini IEF Cell toward the electrode plugs to remove it Remove the Graphite Electrodes from the cell and rinse them with distilled water to remove traces of acrylamide from the previous run The electrodes may be gently wiped with a laboratory tissue if necessary Place the electrodes back into the cell Lightly moisten the graphite electrodes with water Turn the gel with the adsorbed samples upside down and place it directly on top of the electrodes Position the gel carefully the first time as re positioning may damage the gel surface Do not remove the glass plate from the gel gel support backing because its weight insures good contact between the gel and the electrodes Carefully slide the lid back onto the Model 111 Mini IEF Cell Plug the power cables of the cell into a power supply able to generate 500 V constant voltage The cell is now ready to separate samples 10 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 4 2 Run Conditions 1 Focusing is carried out under constant voltage conditions in a stepped fashion Begin focusing at 100 V for 15 minutes Increase voltage to 200 V for 15 minutes Finally increase the voltage to 450 V for an additional 60 minutes Note Step increases of voltage are necessary to prevent overheating and subsequent dehydration of the gel Failure to follow this pro
25. ion between the glass plate and the casting tray as shown in Figure 3 1 a Hold the pipet at a 45 angle and clear the air bubble from the tip b Start the monomer flow at one end of the glass plate and slowly move the pipet to the other spacer c When a liquid front is established across the plate slowly add the remaining monomer from the midpoint of the plate d Control the flow rate to prevent air bubbles If a bubble is trapped remove it by sliding the plate sideways until the bubble is at the edge Remove this bubble a Clear bubble from end of pipet b Start solution at one corner of plate and move it along the edge to form a front Be sure the solution contacts both the plate and tray Avoid trapping air bubbles by releasing the solution slowly c Continue to inject solution keeping the front fairly even to avoid forming bubbles Fig 3 1 Injecting monomer ampholyte solution in casting tray Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 7 Position photopolymerization light such as Bio Rad s catalog number 170 4220 or 170 4242 See Section 9 2 over the tray Any fluorescent desk lamp is a suitable alternative 8 Irradiate the solution for 45 minutes 9 To lift the gel from the casting tray a Lift one corner with a flat spatula inserted between the gel and the casting tray see Figure 3 2 b When air appears under the gel gently
26. ix Dreparatpon enne 5 3 2 Stock Solutions for Polyacrylamide IEF 6 3 3 Reagents for Polyacrylamide Electrofocusing Gels sess 6 3 4 Use of Gel Support Film for Polyacrylamide sees 7 3 5 Casting Polyacrylamide Gels 040000 8 3 6 Sample Dreparapon eene eene nnne nete terrere nennen 9 2 Sample Application eee metere ete iei ations e 10 3 8 Position of Applceapon enne ener eren enne 10 Section 4 Running The Gel 10 4 1 Se tUp Proceedure EHE Reha ia 10 LS MEN tee 11 Section 5 Sample Detection and Gel Storage 2 2 12 2 1 Removing the Gel sinrang eene tette rte petisti eee vens 12 5 2 Band Detection E ETE oa oa 12 Da e ET 13 DA Detection 2 13 5 5 Drying and Preservation 13 Section 6 Agarose Gel Electrofocusing 14 6 1 IntroduG OoD eru eerie tere DE ee eee 14 6 2 Preparing Agarose Gels dO tt ettet ie e 14 6 3 e e EEEE R 16 6 4 Sample Oe 16 6 5 Position of Application e e hee
27. l is not compatible with acetone chlorinated hydrocarbons 1 chloroform or aromatic hydrocarbons 1 toluene benzene To clean the cell components use a mild detergent such as Bio Rad s Cleaning Concentrate catalog number 161 0722 or ethanol Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 1 3 Safety Power to the Model 111 Mini IEF Cell is to be 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 of Bio Rad s power supplies meet this important safety requirement Regardless of which power supply is used the maximum specified operating parameters for the cell are 500VDC maximum voltage limit 5 Watts maximum power limit 50 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 and always turn the power supply off before removing the lid or when working with the cell in anyway IMPORTANT This Bio Rad instrument is designed and certified to meet IEC1010 1 safety standards Certified products are safe to use when operated in accordance with the instruction manual This instrument should not be modified or altered in an
28. lectroendosmosis c Use highest quality acrylamide and Bis to avoid polymerizing acrylic acid into into the gel d Prolonged storage of acrylamide and Bis leads to acrylic acid formation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Problem Cause Solution e Ampholyte contamination e Check ampholyte for bacterial or deterioration contamination Solution will look cloudy Inspect under a microscope Check date of receipt and storage conditions 5 No current power at a Poor gel electrode a Lightly moisten Graphite recommended voltage contact Electrode before applying gel b Electrodes dirty b Remove residue with tissue and water Always clean electrodes after run c Poor electrical contact in c Make sure lid is pushed on unit completely and electrodes are properly seated 7 2 Casting Troubleshooting Polyacrylamide Gels Problem Cause Solution 1 Gel does not adhere to a Incomplete a Useonly high purity backing sticks to casting polymerization acrylamide and Bis Inferior tray monomers are difficult to polymerize Store solutions in amber bottles at 4 for no more than 4 weeks Make fresh catalyst solutions daily Use higher catalyst levels with ampholytes other than Bio Lyte ampholytes Degas longer if gels are polymerized exclusively with APS or TEMED Degas for shorter time if riboflavin is one of the catalysts Pr
29. lift the plate free from the casting tray 10 Flip the plate glass side down onto the casting tray and further irradiate for 15 minutes to eliminate unpolymerized monomer on the gel surface Fig 3 2 Lifting the gel from the casting tray 3 6 Sample Preparation Protein samples for isoelectric focusing must be free of precipitates Substantially salt free samples in typical biochemical buffers are usually tolerated though better results can be obtained with solutions in deionized water 2 ampholytes or 1 glycine Suitable protein solutions may be prepared by dialysis or gel filtration with Bio Spin 6 chromatography columns catalog number 732 6000 10 or 732 6002 25 or with Bio Gel P 6DG desalting gel catalog number 150 0738 Many samples will require the use of urea ethylene glycol non ionic detergents i e Triton X 100 detergent NP 40 Lubrol Wx detergent or octylglucopyranoside or zwitterionic detergents CHAPS CHAPSO Even in the presence of detergents some samples may resist solubility due to salt requirements Only if high salt is an absolute requirement should it be present in a sample and even then substantial distortions and anomalies can be expected Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 7 Sample Application Sample application is most conveniently accomplished using the included Sample Templates 1 Place the template on top o
30. me samples may resist solubility due to salt requirements Only if high salt is an absolute requirement should it be present in a sample and even then substantial distortions and anomalies can be expected 6 4 Sample Application Sample application is most conveniently accomplished using the included Sample Templates 1 Place the template on top of the polymerized gel The colored portion of the template should coincide with the longer dimension of the gel The application position for the sample varies see Section 6 5 but one should allow 1 cm at both the top and bottom of the gel where the gel will contact the electrodes 2 Apply samples using a pipettor capable of delivering between 0 5 ul and 2 ul Volumes above 2 ul are not generally recommended when using the sample template 3 Allow samples to diffuse into the gel for 5 minutes 4 Carefully remove the template from the gel Note For larger amounts and sizes of samples one may custom form application strips from filter paper or use Bio Rad s Sample Application Pieces catalog number 170 4257 Apply the sample to the filter paper and then place the filter paper at the point of application on the gel Allow the sample to diffuse into the gel for 5 minutes The disadvantage of this method is that some proteins may not be completely eluted from the strip to the gel 6 5 Position of Application There are no fixed rules regarding the positioning of the sample on the gel Samples s
31. mply cover the tray with plastic wrap to prevent dehydration of the gel ON To lift the gel from the casting tray a Lift one corner with a flat spatula inserted between the gel and the casting tray Figure 6 1 2 Fig 6 1 Loosening the plate 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 6 3 Sample Preparation Protein samples for isoelectric focusing must be free of precipitates Substantially salt free samples in typical biochemical buffers are usually tolerated though better results can be obtained with solutions in deionized water 2 ampholytes or 1 glycine Suitable protein solutions may be prepared by dialysis or gel filtration with Bio Gel P 6DG desalting gel catalog number 150 0738 A convenient technique for preparing small samples is to load a small amount of hydrated Bio Gel P 6DG gel into a 0 5 ml plastic microcentrifuge tube with a small hole in the bottom Load the sample on top of the gel and place this tube into a 1 5 ml plastic microcentrifuge tube Spin for 5 seconds in a microcentrifuge The sample will be adequately desalted for isoelectric focusing and can be isolated from the bottom of the larger tube Many samples will require the use of urea ethylene glycol non ionic detergents i e Triton X 100 NP 40 Lubrol WX or octylglucopyranoside or zwitterionic detergents CHAPS CHAPSO Even in the presence of detergents so
32. o interfere with gel adhesion to the polyacrylamide support film Increasing the concentration of ammonium persulfate to 0 7 mg ml in the final acrylamide gel solution add 70 ul of 10 ammonium persulfate per 10 ml solution should alleviate the problem Adhesion may also be affected by prolonged soaking in acid solutions such as in staining and destaining Do not soak longer than absolutely necessary to achieve the desired result Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 5 Casting Polyacrylamide Gels Important The Glass Plates and the casting tray must be clean and dry Do not use organic solvents abrasive cleaners or hot water on the casting tray Clean the Glass Plates with soap and water and rinse with deionized water followed by ethanol Wipe the plates dry with lint free paper 1 With the gel support film facing down place the Glass Plate s on the casting tray so that it rests on the spacer bars 2 Prepare the monomer ampholyte solution see Section 3 3 Degas the solution for 5 minutes under vacuum Do not degas longer as a slight amount of is required to catalyze riboflavin 5 phosphate 4 Prepare the catalyst solutions Note Always use freshly prepared persulfate solutions 5 Add the catalyst solutions to the degassed monomer and swirl gently Caution Do not mouth pipet acrylamide solutions Wear gloves Acrylamide is a neurotoxin 6 Pipet the solut
33. oblem Cause Solution Excessive pooling of water at the cathode Agarose only 2 Distortion in gradient where sample is applied 3 Sample streaking 4 pH gradient does not cover expected range a Poor quality agarose b Gel has not been sufficiently blotted dry sufficiently blotted dry prior to run a Too much salt in sample b Sample is applied too near the anode c Sample load excessive d Sample precipitation a Particles in sample b Sample absorbed onto applicator c Precipitation at point of application a Focused too long or use of excessive voltage b Basic gels stored too long c Poor quality acrylamide and Bis d Old acrylamide and Bis stock solutions 19 a Use Bio Rad s Zero Agrose only b Blot all excess liquid from gel prior to run Dialyze against ampholyte1 glycine or water or desalt with Bio Gel P 2 or P 6DG gel b Apply elsewhere on plate minimum of 1 cm from anode c Dilute sample d Spin to remove insolubles or use detergents a Centrifuge sample before application b Change application method c Try a different position Use additives 1 glycine urea non ionic detergents amphoteric detergents a At recommended constant voltage proteins should be focused within 1 5 hours b Use basic gels gt 7 immediately to prevent hydrolysis of acrylamide to acrylic acid which causes e
34. proximately 40 mini IEF gels 9 7 Sample Preperation Catalog Number Description 732 6000 Bio Spin 6 Chromatography Column 10 732 6002 Bio Spin 6 Chromatography Column 25 9 8 Power Supplies Catalog Number Description 165 5056 PowerPac Power Supply 100 VAC 165 5057 PowerPac Power Supply 220 VAC 165 4710 Model 1000 500 Programmable Power Supply 100 120 VAC 50 60 Hz 165 4711 Model 1000 500 Programmable Power Supply 220 240 VAC 50 60 Hz 24 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Life Science Group 2000 Alfred Nobel Drive Hercules California 94547 Telephone 510 741 1000 Fax 510 741 1060 Bio Rad Laboratories Eastern Regional Office 85A Marcus Dr Melville New York 11747 Phone 516 756 2575 Fax 516 756 2594 European Headquarters Bio Rad Laboratories Dreve du S n chal 19 B 1180 Brussels e Phone 02 375 59 70 Fax 02 374 61 62 Australia Bio Rad Laboratories Pty Limited Unit 11 112 118 Talavera Rd Box 371 North Ryde N S W 2113 Phone 02 805 5000 Fax 02 805 1920 Austria Bio Rad Laboratories Ges m b H Auhofstrasse 78D A 1130 Wien e Phone 0222 877 89 01 Fax 0222 876 56 29 Belgium Bio Rad Laboratories S A N V Begoniastraat 5 B 9810 Nazareth Eke Phone 091 85 55 11 Fax 091 85 65 54 Canada Bio Rad Laboratories Canada Ltd 5149 Bradco Boulevard Mississauga Ontario LAW 2 6 e Phone 416 624 0713 Fax
35. re may cause the spacers to separate from the casting tray and may cause the tray to warp 5 Remove a sheet of Gel Support Film for Agarose catalog number 170 2984 from the package 6 Test for hydrophobic side by placing a drop of water on the film The water beads on the hydrophobic side and spreads on the hydrophilic side Pipet few drops of water onto the glass plate d Place the hydrophobic side of the Gel Support Film for Agarose against the plate 9 Roll the Gel Support Film for Agarose flat with a test tube or similar object to force out excess water and air bubbles 10 Remove the casting tray from the oven and place it on a level surface 11 Heat a glass pipet with hot water and pipet 4 ml of the warm agarose solution in a bead across the width of the casting tray 12 Carefully lower the Glass Plate with the gel support film side down onto the agarose Allow the agarose to spread completely underneath the glass plate Some agarose may extend outside the glass plate 13 If there are any areas under the Glass Plate not filled with agarose simply tilt the casting tray and the agarose should fill in these areas quickly 14 If there are any air bubbles trapped tapping on the Glass Plate should remove them 15 The gel will solidify within 10 15 minutes At this point let the gel age at 4 C refrigerate for a minimum of 4 hours preferably overnight The gels may be left in the casting tray Si
36. roduction to Isoelectric Focusing 2 1 The Electrofocusing Principle Conventional electrophoresis separates proteins and other charged molecules by electrophoretically driven migration through a sieving matrix that is buffered at a constant pH Each component of the mixture assumes its own characteristic velocity based on molecular size and surface charge This velocity is constant throughout the electrophoresis experiment and is counteracted by diffusion which tends to broaden the bands There is no tendency toward equilibrium in conventional electrophoresis and the protein bands will run off the gel if the electrical field is not interrupted On the other hand electrofocusing separates proteins on the basis of surface charge alone as a function of pH The separation is done in a non sieving medium sucrose density gradient agarose or polyacrylamide gel in the presence of carrier ampholytes which establish a pH gradient increasing from the anode to the cathode Since a protein contains both positive amines and negative carboxyl charge bearing groups the net charge of the protein will vary as a function of pH A pH gradient is established concomitantly with protein separation As the protein migrates into an acidic region of the gel it will gain positive charge via protonation of the carboxylic and amino groups some point the overall positive charge will cause the protein to migrate away from the anode to a more basic region of
37. t proteins in ug quantities Either method produces acceptable results however Method B is significantly easier to use and in some cases will produce a sharper pattern For more sensitive detection down to ng levels of protein the Bio Rad Silver Stain catalog number 161 0443 is recommended The cupric sulfate in the staining and destaining solutions effectively eliminates any background staining due to the presence of ampholytes Fixing and Staining Method A Fixative 4 sulfosalicylic acid 12 5 trichloroacetic acid 30 methanol Immerse gels in this solution for 30 minutes Method A Stain 27 isopropanol or ethanol 10 acetic acid 0 04 Coomassie brilliant blue R 250 0 5 CuSO4 0 05 crocein scarlet optional Dissolve the CuSO4 in water before adding the alcohol Either dissolve the dye in alcohol or add it to the solution at the end Immerse the gel in the stain for approximately 1 2 hours 12 Method B Fixative Not necessary Method B Stain Same as Method A but with 0 05 crocein scarlet Crocein scarlet a highly soluble dye which rapidly binds to protein is included to assure rapid fixation of the bands This procedure is inadequate if Coomassie brilliant blue R 250 is used alone Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 5 3 Destaining For both Method A and Method First destaining solution 12 isopropanol or
38. the gel As the protein enters a more basic environment it will lose positive charge and gain negative charge via ammonium and carboxylic acid group deprotonation and consequently will migrate away from the cathode Eventually the protein reaches a position in the pH gradient where its net charge is zero defined as its pI or isoelectric point At that point the electrophoretic mobility is zero Migration will cease and a concentration equilibrium of the focused protein is established 2 2 Carrier Ampholytes Carrier ampholytes are complex mixtures of amphoteric buffers that form a smooth pH gradient in an applied electrical field During electrofocusing these buffers stack according to their individual pls across the gel producing a linear gradient In order for the gradient to appear smooth and continuous a large number of these buffering components must be present This is also a requirement for separating a complex mixture of proteins Bio Lyte ampholytes are derivatized low molecular weight amines that are electrophoretically separated and reblended to give smooth and reproducible gradients Narrow range Bio Lyte ampholytes are produced and tested so that under normal circumstances no additional blending or fortification will be necessary to achieve the desired shallow gradient Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 2 3 Choice of Support Matrix The electrofocus
39. tical isoelectric focusing applications The gel is run upside down directly contacting the electrodes to eliminate the need for electrode buffers and wicks There is no need for active cooling during the focusing process Condensation within the cell is not a problem because the gel is run upside down and therefore the condensate cannot fall onto the gel The compact Model 111 Mini IEF Cell contains two graphite electrodes with an inter electrode distance of 5 cm The graphite electrodes are removable for easy cleaning A maximum applied voltage of 450 volts is sufficient to yield tight bands with excellent separation within 90 minutes The Model 111 Mini IEF Cell makes use of ultra thin 0 4 mm gels which provide better heat dissipation than thicker gels Casting these gels is simple with the casting tray and gel support film provided with the cell Acrylamide and agarose gels are both cast using the same casting tray 1 2 Specifications Construction Outer chamber Cell lid Casting tray Electrodes Sample templates Shipping weight Overall size Casting tray Gel size Glass plate size Voltage power limit Casting tray temperature limit Fabricated acrylic Polycarbonate Fabricated acrylic High purity graphite 0 95 cm diameter Polyvinyl chloride 1 8 kg 24 6 1 x 11 4 x 4 8 cm h 20 3 I x 13 9 x 2 3 cm h 125 x 65 I x 0 4 mm t 125 x 65 mm 500 VDC 5W 60 Note The Model 111 Mini IEF Cel
40. y way Alteration of this instrument will Void the manufacturer s warranty Void the IEC1010 1 safety certification Create a potential safety hazard 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 EC 1010 1 is an internationally accepted electrical safety standard for laboratory instruments Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 1 4 Model 111 Mini IEF Cell Components Fig 1 1 Model 111 Mini IEF Cell Outer chamber 1 sliding interlocking lid with power cables 2 graphite electrodes 3 casting tray 4 glass plates 5 sample templates 6 gel support film 7 and 5 ml Bio Lyte 3 10 ampholyte 8 1 5 Capillary Thin Layer Gel Casting Tray The Capillary Thin Layer Gel Casting Tray provides the fastest and easiest method for casting electrofocusing gels The casting tray consists of an acrylic plate with precisely defined spacers of 0 4 mm thickness as shown in Figure 1 2 The acrylic surface imparts a slight inhibitory effect on acrylamide polymerization eliminating sticking and tearing of the gel Glass plate Spacer rails Fig 1 2 Casting tray Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 2 Int
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