Criterion™ Blotter Instruction Manual - Bio-Rad
Contents
1. 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 Catalog Number Date of Delivery Serial Number Invoice Number Purchase Order No 19 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 610 741 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 69 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 63981 12 113 114 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 642. sessesessssesossesossssesossesossesosoesosossesossesossee 12 Protein Blotting Membranes ccccccceseesesseseseeseseeceeeseeeeaeeeeseeeeseeseecaeereaeeeeseeaeas 12 DNA and RNA Blotting Membranes ccccccecesseseeseseeseseeseseeeeseeseseeseeeeaeeeeseeeees 13 Troubleshooting Guide sesossesossssesossesossesossesosossesossesossesossssosossesossesossee 14 Electrophoretic Transfer cccccceccssesesseseeseseeseseeseseeceseeseeecaeseeseeeeseeaesesaeeeeseeeeseeeeas 14 Maintenance cccsccsscsscssscssscsscsscssscsscesscsssssscessssssssecessessesssssssessessesesoes 16 Product nformation cccccsssssssssccsscsssssssssscsssssssssscesssssessssescsssesseees 16 References OENE SEESE EEE E E E E 17 Warranty sssissccssasossessccaceessasssnecessossesseasenssease scesseascussvacensessosscusteasonsensecsesesotes 19 Section 1 General information 1 1 Introduction The Criterion Blotter is an electrophoretic transfer cell designed for use with Criterion precast gels as well as hand cast gels The Criterion Blotter cell is available with standard wire platinum electrodes or with plate electrodes which consist of a platinum coated titani um anode and a stainless steel cathose Transfers are performed with either set of electrodes positioned 4 3 cm apart using one or two gel holder cassettes positioned between the elec trodes This allows generation of ahigh intensity electrical field for and efficient transfer w
3. handling and for reprobing Immun Blot PVDF 0 2 um 150 160 High mechanical strength and chemical stability used for immune detection western blotting low background to signal ratio enhanced binding in the presence of SDS Must be wet in 100 MeOH before equilibration in buffer Sequi Blot PVDF 0 2 um 170 200 High mechanical strength and chemical stability used for protein sequencing enhanced binding in the presence of SDS Must be wet in 100 MeOH before equilibration in buffer 13 Section 6 Troubleshooting Guide 6 1 Electrophoretic Transfer Poor or no electrophoretic transfer as detected by staining the gel 1 5 Transfer apparatus is assembled incorrectly and the proteins are moving in the wrong direction The gel membrane sandwich may be assembled in the wrong order or the cassette is inserted in the tank with the incorrect orientation Check the polarity of the connections to the power supply Detection system is not working or not sensitive enough e Include proper positive and negative control antigen lanes to test for detection kit sensitivity Consult kit manual Transfer time is too short e Increase the transfer time Charge to mass ratio is incorrect Native transfers e Try a more basic or acidic transfer buffer to increase protein mobility Proteins near their isoelectric point at the pH of the buffer will transfer poorly It has been suggested that buffer pH should be 2 pH units high
4. 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 100 MeOH prior to equilibration in aqueous transfer buffer Follow the directions in the product insert 3 The gel electrophoresis may be at fault e Artifacts of electrophoresis may be produced by poor polymerization inappropriate running conditions contaminated buffers sample overload etc Consult your electrophoresis manual for more details Gel cassette pattern transferred to blot 1 Contaminated or worn fiber pads are used e Replace the fiber pads or thoroughly clean the contaminated pads 2 The transfer buffer is contaminated e Make fresh solutions Poor Binding to the Membrane Nitrocellulose 1 20 methanol in the transfer buffer is optimal for protein binding e Make sure the buffer contains the proper amount of methanol 2 Proteins may be transferring through the nitrocellulose e Use PVDF or nylon higher binding capacities or 0 2 um nitrocellulose smaller pore size Decrease the voltage 3 Proteins lt 15 000 daltons may show diminished binding to 0 45 um nitrocellulose or may be washed from the membrane during assays e Use PVDF or nylon membrane which have higher binding capacities e Use only Tween 20 de
5. somewhat more difficult to quantitatively transfer large amounts of DNA used in genomic blots The following tactics should be considered for optimizing elution in such transfers 1 Alter 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 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 PVDF Membrane Bio Rad offers PVDF Polyvinylidene difluoride membranes 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 acid base and in the presence of organic solvents 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 retention during sequencing manipulations enhances the likelihood of obtaining information from rare low abundance proteins by increased initial coupling and higher repetitive yields In addition PVDF membrane exhibits better b
6. the back large compartment of the tray Open the cassette so that the red side with the handle is vertical anode and the black side cathode is laying horizontal and submerged in transfer buffer d Place a fiber pad on top of the black side of the cassette submerged in buffer Push on the fiber pad with gloved finger tips to thoroughly soak the pad e Place a piece of filter paper on top of the fiber pad it will wet immediately f Gently place the pre equilibrated gel on top of the filter paper Use the roller to remove any air bubbles that may be trapped underneath the gel g Take the membrane from the front compartment and place it on top of the gel taking care not to trap any air bubbles The membrane should not be moved or adjusted once it touches the gel because this can cause data ghost prints and artifacts If you feel that you must adjust the membrane placement use a fresh pre wetted membrane Use the roller to roll out bubbles see figure h Place a piece of filter paper on top of the membrane Run the roller gently over the top of the filter paper to remove any air bubbles trapped in the sandwich i Wet a second fiber pad in the front compartment of the tray where the membrane was soaking again using finger tips to completely saturate the pad with transfer buffer Then place the wet fiber pad on top of the second filter paper j Lower the clamp side of the cassette and lock in the closed position see figure No
7. 12 195 1981 Legocki R P and Verma D P S Anal Biochem 111 385 1981 Lin W and Kasamatsu H Anal Biochem 128 302 1983 Anderson N L Nance S L Pearson T W and Anderson N G Electrophoresis 3 135 1982 McLellan T and Pamshaw J A M Biochem Genetics 19 647 1981 Gibson W Anal Biochem 118 1 1981 Howe J G and Hershey J W B J Biol Chem 2566 12836 1981 Erickson P G Minier L N and Lasher P S J Immun Meth 51 241 1982 Tsang V C W Peralta J M and Simons A R Meth Enzymol 92 377 1983 Gershoni J M and Palade G E Anal Biochem 124 396 1982 Gershoni J M and Palade G E Anal Biochem 131 1 1983 Symington J Two Dimensional Gel Electrophoresis of Proteins Methods and Applications Celis J E and Bravo R eds Academic Press N Y 1983 Andrews A T Electrophoresis Theory techniques and biochemical and clinical application 2nd ed Clarendon Press Oxford 1986 Beisiegel V Electrophoresis 7 1 1986 Bio Rad Laboratories unpublished Gershoni J M in Advances in Electrophoresis Vol 1 Chrambach A Dunn M J and Radola B J eds VCH Weinheim in press Gershoni J M in Methods of Biochemical Analysis Vol 33 Glick D ed Wiley New York in press Bjerrum O J and Schafer Nielsen C Analytical Electrophoresis M J Dunn ed VCH Weinheim p 315
8. 61 0733 1L bottles or catalog number 161 0770 5L cube 900 ml of dd H O b TAE Tris Acetate EDTA 40 mM Tris Acetate 1 mM EDTA 20 ml of 50X TAE buffer catalog number 161 0743 1L bottles or catalog number 161 0773 5L cube 980 ml of dd H O 10 Section 4 Strategies for Optimizing Electro Elution 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 Failure of molecules to bind efficiently to the membrane caused by poor gel membrane contact is often confused with inefficient elution Poor contact is usually due to excess moisture in the gel membrane interface Proper technique and the use of a test tube or roller should assure good contact Proper selection of filter paper spacers will help assure good compression Gel and membrane equilibration in transfer buffer for at least 15 minutes prior to transfer will help prevent shrinking or swelling of either component during transfer and will 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 30 minutes to as long as overnight Remember all overnight applications should be performed at 30 50 volts to minimize heating problems For long transfers at elevated voltages use th
9. 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 7095 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 4006190 Rev B
10. Criterion Blotter Instruction Manual Catalog Numbers 170 4070 170 4071 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 Section 1 1 1 1 2 1 3 Section 2 2 1 22 2 3 Section 3 3 1 3 2 Section 4 4 1 4 2 Section 5 5 1 5 2 Section 6 6 1 Section 7 Section 8 Section 9 Section 10 Table of Contents Page General Information eseserororocoesesesesesesesesorororocosoesesesesesesosororororoeoesesesesesesee 1 Fntrod ucti on Manea e a a e a aai 1 Specifications nininini a a a e e e iian 2 SITAN acta heh ate te EEA AEE E EEA E 2 Set Up and Basic Operation sessesessesesossesossesosossesossosossesossesossssosossesosseso 3 Preparation for BIOtINE seai ninne niea iii 3 Begin B ENIES E AEA 5 Acidic Transfers monn a e e a a a aR dite teieni 6 Transfer Conditions cccsccsssssssssscsssssssssscsssssscsssssscesscsssssssessessssssseses 6 General Guidelines to Transfer Buffers and Running Conditions cece 6 Notes on Electrophoretic Transfer Conditions ccceseseseeseseeseseeseseeseeeeseeeeseeeeaes 8 Strategies For Optimizing Electro Elution scscssssssscssssssssscsssoees 11 Optimizing Protein Transfer cccececcesceceseeseseeseseeceseeseecseeceseeecseeseseeaeeeeaeeeeseeaees 11 Optimizing DNA and RNA Transfer c ccccececceesseseseeseeseseeseseeeeseesesecaeereseeeeaeeeeas 12 Choice of Blotting Membranes
11. DC voltage output floats with respect to ground All 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 300 VDC Maximum voltage limit 200 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 any way The Criterion Blotter 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 Criterion Blotter 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
12. Dunn S D Anal Biochem 157 144 1986 Zeta Probe Instruction Manual Bio Rad Laboratories 1988 Polvino W J Saravis C A Sampson C E and Cook R B Electrophoresis 4 368 1983 Bio Rad Laboratories Biotin Blot Total Protein Stain Instruction Manual 1985 LaRochelle W J and Froehner S C J Immunol Meth 92 65 1986 Szewcyzyk B and Kozloff L M Anal Biochem 150 403 1985 Perides G Plagens U and Traub P Anal Biochem 152 94 1986 Scotch Brite is a registered trademark of 3M Company Gel Bond is a trademark of FMC Mylar is a registered trademark of E I DuPont de Nemours Co Coomassie is a trademark of ICI 18 Section 10 Warranty The Criterion Blotter 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 or an authorized agent 3 Use of spare parts supplied by anyone other than Bio Rad Laboratories 4 Damage caused by deliberate or accidental misuse 5 Corrosion due to use of improper solvent or sample Use with chlorinated hydrocarbons e g chloroform aromatic hydrocarbons e g toluene benzene or acetone
13. References 1 D Southern E M J Mol Biol 98 503 1975 Alwine J C Kemp D J Parker B A Reiser J Renart j Stark G R and Wahl G W Methods Enzymol 68 220 1979 Thomas P S Proc Nat Acad Sci 77 5201 1980 Seed B Nuc Acids Res 10 1799 1982 Renart J Peiser J and Stark G R Proc Nat Acad Sci 76 3116 1979 Bowen P Steinberg J Laemmli U K and Weintraub H Nuc Acids Res 8 7 1980 Towbin H Staehelin T and Gordon J Proc Nat Acad Sci 76 4350 1970 Bittner M Kupferer P and Morris C R Anal Biochem 102 459 1980 Stellwag E J and Dahlberg A E Nuc Acids Res 8 299 1980 Kutateladze T V Axelrod B D Gorbulev V G Belzhelarshaya S N and Vartikyan R M Anal Biochem 100 129 1979 Peudelhuber T L Ball D J Davis A H and Garrard W J Nuc Acids Res 10 1311 1982 Danner D B Anal Biochem 125 139 1982 Bio Rad Technical Bulletin 1110 Zeta Probe Blotting Membranes 1982 17 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 39 40 41 42 Holland L J and Wangh L H Nuc Acids Res 10 3283 1983 Syminton J Green M and Brackmann K Proc Nat Acad Sci 78 177 1981 Reiser J and Wardale J Eur J Biochem 114 569 1981 Burnette W N Anal Biochem 1
14. agent Section 2 Set Up and Basic Operation Criterion Blotter Cell Assembly 2 1 Preparation for Blotting Is 2 3 4 Freeze ice block prior to preparation of blot assembly Prepare the transfer buffer See Section 3 3 for buffer formulation Using buffer pre chilled to 4 C will improve heat dissipation Cut membrane and the filter paper to the dimensions of the gel if necessary Equilibrate gel in transfer buffer for 15 minutes Note Always wear clean gloves when handling the transfer cell membranes filter paper or gels to prevent contamination See Sectioin 3 2 for details 5 Set up transfer apparatus e Fill the Criterion Blotter tank with transfer buffer to about 50 of the fill volume e Place a magnetic stir bar inside the tank 0 8 10 mm e Place the ice block in the ice block pocket in the back of the cell Flip down the lever to hold the ice block down e Alternatively the optional cooling coil can be used Connect it to an appropriate recirculating water chiller and place it in the grooves in the back of the tank New Figure Fiber pad Blot absorbant paper Membrane Gel Blot absorbant paper Fiber pad 4 Set up the gel membrane sandwich a Pour chilled transfer buffer into each compartment of the gel blot assembly tray b Place the membrane nitrocellulose PVDF etc in the front small compartment of the tray Let it soak while you set up steps c g c Place the cassette in
15. anol 10 Methanol 15 Ethanol Criterion Blotter 100 V 100 V 100 V with plate electrodes 30 minutes 30 minutes 30 minutes Criterion Blotter 100 V 100 V 100 V with wire electrodes 60 minutes 30 minutes 60 minutes We find nearly undetectable levels of proteins remaining in the gel but some protein blow through is observed under this condition PVDF is suggested for transfer of low molecular weight proteins Table 3 3 Native Gels These conditions were determined empirically using 12 5 Tris HCl Criterion gels and four native protein samples cytochrome C pI 9 6 lentil lectin pI 8 2 8 0 7 8 carbonic anhydrous pI 6 0 and glucose oxidase pI 4 5 Buffer 1X Tris Glycine see Section 3 3 Buffer formulation The transfer of proteins from Native gels will depend on the size and pI of the protein relative to the pH of the buffer used during transfer If the pI of the protein is greater than the pH of the transfer buffer the protein will carry a positive charge and will travel toward the negative electrode cathode The voltage suggested is a starting point The transfer time will need to be determined empirically for your protein of interest Criterion Blotter Overnight 12 hrs 30 minutes with plate electrodes Max 10 V 50 V Max 50 mA 750 950 mA Criterion Blotter Overnight 12 hrs 60 minutes with wire electrodes Max 10 V 50 V Max 50 mA 300 500 mA Note The power supply should be set on these maximum set
16. atures at the end of the transfer are acceptable Where precise temperature control is required or transfers runs longer than 1 hour at high voltage conditions the optional cooling coil connected to a refrigerated recirculating bath should be used Placing the Criterion Blotter cell in the cold room is an inadequate means of controlling transfer buffer temperature The tank of the Criterion Blotter cell is an effective thermal insulator thus limiting the efficient dissipation of heat 6 Use of a stir bar during transfer For all blotting applications a stir bar must be placed inside the Criterion Blotter cell so that the transfer buffer is stirred during the course of the experiment This will help to maintain uniform conductivity and temperature distribution during electrophoretic transfer Use of a stir bar prevents the appearance of tiny bubbles on blots as well 7 Transfer buffer pH Do not adjust the pH of transfer buffers unless specifically indicated Adjustments of the pH of transfer buffers when not indicated will result in increased buffer conductivity This is manifested by a higher than expected initial current output possible heating problems and a decreased resistance 8 Transfer buffer recommendations Use only high quality reagent grade methanol Contaminated methanol can result in increased transfer buffer conductivity as well as poor transfer of macromolecules Reuse of transfer buffers is not advised since these buffe
17. ct 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 nitrocellulose membranes e Eliminate alcohol from the transfer buffer Alcohol in the transfer buffer is required for binding of SDS proteins to nitrocellulose Elimination of alcohol results in increased transfer efficiency but diminishes binding to nitrocellulose Transfer efficiency is decreased because alcohol causes gel pores to contract resulting in fixation of large molecular weight proteins within the gel matrix Use of PVDF membrane for SDS protein transfers may reduce the alcohol requirement and constitutes a logical strategy for analysis of high molecular weight or difficult to transfer proteins 27 11 5 Alter membrane type As mentioned in 4e PVDF membrane allows transfer in reduced alcohol see Section 5 1 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 for proteins that will be sequenced or delivered to mass spec 6 Increase pore size or decrease T so that proteins will not be trapped inside small pores 4 2 Optimizing DNA and RNA Transfer Problems with elution of nucleic acids can be solved by altering the gel percentage It may be
18. e Super Cooling Coil Increase the field strength Initial controls should be performed to evaluate the efficiency of increasing the V cm as well as its effects on 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 Vary buffer type and pH a Reduce 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 charge to mass ratio It appears that alcohols present in SDS transfer buffer strip SDS from proteins Basic proteins in Tris glycine methanol buffer at pH 8 3 may assume a state near isoelectric neutrality and thus transfer poorly For example lysozyme exhibits this behavior 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 c Different buffer types at similar V cm may yield different efficiencies Generally Tris buffers allow more efficient transfer than acetate or phosphate buffers d Addition of 0 01 to SDS detergent to Tris glycine methanol buffer has been reported to increase transfer efficiency 2 SDS however increases relative current power and heating Also temperatures below 10 C may precipitate the SDS so the starting buffer temperature will be higher SDS may also affe
19. e gel to adjust to its final size prior to electrophoretic transfer If the isoelectric point of the protein is close to the pH of the buffer the protein may not leave the gel If the isoelectric point of the protein is 2 pH units below the pH of the buffer the protein will be negatively charged and will migrate towards the positive electrode anode 7 2 Current limits The PowerPac 200 Power Supply is capable of a 200 watt output Unless a current limit is set uncontrolled conductivity changes may result in full power being delivered to the Criterion Blotter cell The gel holder and electrode cards may warp and the transfer buffer may heat up further increasing conductivity This would result in a potential safety hazard Refer to the PowerPac 200 Power Supply Instruction Manual for procedures to set current limits and run times 3 Polarity of transfer Do not reverse polarity with 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 abrasive cleanser to remove the rust Damage to electrode plates is not covered under warranty 4 Heat dissipation Two methods of heat dissipation are available The efficient transfer and high intensity field of the Criterion Blotter requires some method of heat dissipation The sealed ice block cooling system is adequate for transfers of less than 1 hour where slightly elevated temper
20. ee the power guidelines for specific applications in Section 3 e Remake the buffer or alter the voltage increase or decrease Try changing the intensity of blotting wire vs plate electrodes Immune Specific Detection Overall high background low signal or lack of development of positive control e Consult instructions for immune detection kit or reagents Total Protein Detection Consult user manual for stain or detection kit Section 7 Maintenance Cleaning Use mild soap and warm water to clean the electrodes cassettes and buffer tank Use special care when cleaning the electrode cards or plate electrodes Avoid stretching or breaking the platinum wires Avoid scratching or marring the platinum plate Do not use abra sives or strong detergents The cathode plate stainless steel can be cleaned with a mild abra sive to remove salt that may be deposited during normal operation Rinse the fiber pads under hot water and then in distilled deionized water Chemical compatibility The Criterion Blotter 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 Section 8 Product Information Catalog Number Product Description 170 4070 Criterion Blotter Plate electrodes includes Cell assembled with plate electrodes lid with cables 2 Criterion gel holder cassettes filter paper pack f
21. er or lower than the pI 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 e Reduction of methanol results in increased transfer efficiency of proteins from the gel but it also diminishes binding to nitrocellulose and PVDF Protein is precipitating in the gel 1 Try using SDS in the transfer buffer SDS can increase transfer efficiency but can also reduce binding efficiency to nitrocellulose and affect reactivity of proteins with some monoclonal antibodies 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 e Use the included roller test tube or pipet as a rolling pin and roll over the membrane carefully in both directions until air bubbles or excess buffer is removed from between gel and membrane and complete contact is established e Use thicker filter paper in the gel membrane sandwich e Replace the fiber pads Pads will compress with time and will not hold the membrane to the gel 14 2 The membrane is not properly wet or has dried out e White spots on the nitrocellulose membrane indicate dry areas where protein will not bind If wetting does
22. hen used in combination with the PowerPac 200 Power Supply Cooling is required for temper ature control and can be achieved with the sealed ice block included with the cell or with the optional cooling coil The Criterion Gel Blot Assembly Tray provides for lab tidy assembly of gel blot sandwiches and gel soaking while minimizing the possibility of incorrect sandwich assembly The foller is useful to ensure proper contact and removal of trapped bubbles dur ing sandwich assembly 1 2 Specifications Criterion Blotter tank Overall dimensions Material Buffer requirement Electrodes Electrode Dimension Material Support card Anode plate Cathode plate Wire electrodes Distance Anode to Cathode Cassettes Cassette dimension Material Maximum gel size Gel Capacity Gel Blot Assembly tray Material Overall dimensions Assembly compartment Soaking compartment 11 8 x 21 8 x 15cm Molded polysulfone 1 3 liters 9 45 x 13 84 cm Red or black molded polysulfone Platinum coated titanium Stainless steel Platinum wire 4 3 cm 11 4 x 16 5 cm Red or black molded polysulfone 9 4 x 15 cm 2 Criterion or 4 Ready Gel precast gels Molded polycarbonate 17 3 x 32 5 x 5 7 cm 14 3 x 17 3 x 3 2 cm 12 1 x 17 3 x 3 2 cm 1 3 Safety The instrument is for research purposes only Power to the Criterion Blotter cell is supplied by an external DC voltage power supply This power supply must be ground isolated in such a way that the
23. iber pad pack gel blot assembly tray sealed ice cooling unit manual roller 170 4071 Criterion Blotter Wire electrodes includes Cell assembled with wire electrodes lid with cables 2 Criterion gel holder cassettes filter paper pack fiber pad pack gel blot assembly tray sealed ice cooling unit manual roller 16 Catalog Number Product Description 170 4072 Criterion Blotter 170 4070 with Plate electrodes and PowerPac 200 Power Supply 110 120 V 170 4073 Criterion Blotter 170 4070 with plate electrodes and PowerPac 200 Power Supply 220 240 V 170 4074 Criterion Blotter 170 4071 with wire electrodes and PowerPac 200 Power Supply 110 120 V 170 4075 Criterion Blotter 170 4071 with wire electrodes and PowerPac 200 Power Supply 220 240 V 170 4076 Optional Criterion Blotter Cooling Coil 165 5052 PowerPac 200 Power Supply 110 120 V 165 5053 PowerPac 200 Power Supply 220 240 V Criterion Blotter Cell Accessories 170 4080 Criterion Gel Holder Cassettes 1 170 4081 Criterion Blotter Platinum Anode Plate Electrode 170 4082 Criterion Blotter Stainless Steel Cathode Plate Electrode 170 4083 Criterion Blotter Standard Wire Electrode Card anode 170 4084 Criterion Blotter Standard Wire Electrode Card cathode 170 4085 Filter Paper 9 5 x 15 2 cm 50 170 4086 Fiber Pads 9 5 x 15 2 cm 4 170 4087 Sealed Ice Cooling Unit 2 170 4089 Criterion Gel Blot Assembly Tray 165 1279 Roller Section 9
24. inding efficiency of blotted material in the presence of SDS in the transfer buffer PVDF must first be wetted in 100 MeOH Nitrocellulose Membrane Nitrocellulose membranes have been used extensively for protein binding and detection 73427 They can be easily stained for total protein by a dye stain Amido Black Coomassie Blue Ponceau S Fast Green FCF etc or the more sensitive Colloidal Gold Total Protein Stain and also allow either RIA FIA or EIA Nonspecific protein binding sites are easily and rapidly blocked avoiding subsequent background problems No pre activation is required Low molecular weight proteins especially lt 20 000 daltons may be lost during post transfer washes thus limiting detection sensitivity Smaller pore size nitrocellulose membrane 0 2 um has been shown to be effective in eliminating this loss Large proteins 2 100 000 daltons denatured by SDS may transfer poorly due to the addition of alcohol to the transfer buffer Alcohol increases binding of SDS proteins to nitrocellulose but decreases 12 pore sizes in the gel Elimination of alcohol from SDS protein transfers results in considerably diminished binding Adding SDS up to 0 1 to the transfer buffer increases the transfer efficiency of proteins but reduces the amount of binding to the membrane Also SDS increases the conductivity of the buffer and the heat generated during transfer 5 2 DNA and RNA Blotting Membrane Zeta Probe Nylo
25. is 14 4 g glycine in 600 ml of dd H O add distilled deionized water dd H O to 900 ml add 100 ml Methanol pH can be adjusted for CAPS buffer CAPS Buffer with 20 Methanol 10 mM CAPS 3 cyclohexylamino 1 propane sulfonic acid 20 v v methanol pH 11 From dry reagents Mix 2 21g CAPS in 600 ml of dd H O ADJUST the pH to 11 0 with NaOH add distilled deionized water dd H O to 800 ml add 200 ml Methanol CAPS Buffer with 10 Methanol 10 mM CAPS 3 cyclohexylamino 1 propane sulfonic acid 10 v v methanol pH 11 From dry reagents Mix 2 21g CAPS in 600 ml of dd H O ADJUST the pH to 11 0 with NaOH add distilled deionized water dd H O to 900 ml add 100 ml Methanol CAPS Buffer with 15 Ethanol 10 mM CAPS 3 cyclohexylamino 1 propane sulfonic acid 15 v v ethanol pH 11 From dry reagents Mix 2 21g CAPS in 600 ml of dd H O ADJUST the pH to 11 0 with NaOH add distilled deionized water dd H O to 850 ml add 150 ml Ethanol Native gels 25 mM Tris 192 mM glycine pH 8 3 a Use Premixed buffer concentrate solution 100 ml of 10X Tris Glycine buffer catalog number 161 0734 1L bottles or catalog number 161 0757 5L cube 900 ml of dd H O b From dry reagents Add 3 03 g Tris 14 4 g glycine to distilled deionized water dd H O then mix and add dd H O to a final volume of 1 liter Nucleic Acid gels a TBE Tris Borate EDTA 89 mM Tris borate 2 mM EDTA pH 8 3 100 ml of 10X TBE buffer catalog number 1
26. lines to Transfer Buffers and Running Conditions Tables 3 1 to 3 4 provide guidelines for power conditions using different buffers Power conditions are provided for various run times The transfer times will need to be increased for gradient gels or they may be decreased if your protein of interest is low molecular weight and transfers quickly The suggested conditions give at least 95 transfer of the sample proteins visible in the gel by Silver Stain Plus catalog number 161 0449 sensitive to ng level and in the blot by Colloidal Gold catalog number 170 6517 sensitive to 4 ng Table 3 1 SDS PAGE Gels These conditions were determined empirically using 12 5 Tris HCl Criterion gels and total proteins from E coli lysates Buffer 1X Tris Glycine see section 3 3 Buffer formulation 20 Methanol 10 Methanol 15 Ethanol Criterion Blotter 100 V 100 V Not recommended with plate electrodes 30 minutes 30 minutes Criterion Blotter 100 V 100 V Not recommended with wire electrodes 60 minutes 30 minutes Our tests show only 60 transfer of E Coli proteins in 1 hour at 100V The ethanol buffer might work if longer transfers are acceptable or if your target protein transfers under this condition Table 3 2 SDS PAGE Gels CAPS based buffers These conditions were determined empirically using 12 5 Tris HCl Criterion gels and total proteins from E coli lysates Buffer 10mM CAPS buffer see Section 3 3 Buffer formulation 20 Meth
27. n Membrane Nitrocellulose is not a suitable medium for electrophoretic transfer of nucleic acids as high concentrations of salt 10 x SSC are required for efficient binding Molecules 500 bp are not bound at all even at high salt Low resistance results when an electric current is passed through a solution of high salt This causes potentially damaging high currents and power at very low voltages Since V cm is the eluting force inefficient transfer occurs under conditions required for proper binding Zeta Probe membrane allows efficient binding of all sizes of single stranded DNA and RNA in the presence of low ionic strength buffers Zeta Probe membrane is an ideal alternative to nitrocellulose for the analysis of nucleic acids Binding is more stable through post transfer washes and reprobing may be performed as many as 10 times Table 5 1 Guide to Protein Blotting Membranes A variety of blotting membranes is available for immunoblotting 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 Binding Capacity Membrane Pore Size ug em2 Notes 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 Nitrocellulose 0 2 um increased strength for easier
28. rs have most likely lost their ability to maintain a stable solution pH during transfer Dilution of transfer buffers below their recommended levels is also not advised since this will decrease their buffering capacity 9 Voltage limits Do not increase voltage settings beyond those indicated in Tables 3 1 3 4 for overnight operation Initial current readings 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 for your application and higher voltages are necessary transfer times must also be adjusted to a shorter length Failure to do so may result in a potential safety hazard 10 These variables will change total resistance and thus the current readings e Alterations in buffer make up i e addition of SDS or changes in ion concentration due to 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 properly equilibrated e Number of gels current increases slightly as the number of gels increases Transfer temperature current increases when temperature increases e Time in transfer at which reading was taken current normally increases as the buffering capacity diminishes with progress of the run 3 3 Buffer Formulation All formulas provided below are for a total volume of 1 liter of buffer 1 3 liters of buffer are required for the Criterion Blo
29. te New fiber pads will create a tight fit when the cassette is closed Subsequent use will make the pads more compressible 2 2 Begin Transfer a Move the locked cassette into the groove in the blotter tank aligning the red side of the card with the red electrode Make sure the magnetic stirrer is free to move see figure b After both cassettes are in place add the remaining transfer buffer to the fill level marked on the tank c Puton the lid plug the cables into the power supply and run the blot Refer to Section 3 for run times with various buffers d Transfer at a constant voltage using the Bio Rad PowerPac 200 which has current capacity of 2A Upon completion of the run disassemble the blotting sandwich and remove the membrane for development Clean the cell fiber pads and cassettes with multiple rinses of deionized water 5 2 3 Acidic Transfers If transferring under acidic buffer pH switch the gel and membrane in the set up instructions or simply reverse the orientation of the gel assembly or reverse the cassette when inserted in to the tank This will place the membrane on the cathode side of the gel Under acidic conditions proteins will transfer in the opposite direction going toward the negative cathode Do not reverse the electrodes themselves or plug the banana plugs into the reverse poles This will cause irreversible damage to the plate electrodes Section 3 Transfer Conditions 3 1 General Guide
30. tergent in the wash and antibody incubation steps Reduce or eliminate the more stringent washing conditions 4 SDS in the transfer buffer will reduce binding efficiency of proteins e 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 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 Poor Binding to the Membrane PVDF 1 The membrane may not be completely wet e Because of the hydrophobic nature of PVDF the membrane must be prewet in 100 MeOH prior to equilibration in aqueous transfer buffer Follow the directions in the product insert 15 2 The membrane may have been allowed to dry during handling e A completely wet membrane has a gray translucent appearance White spots will form on the surface of the membrane as it dries Since proteins will not bind to the dry spots rewet the membrane with methanol and re equilibrate in transfer buffer Power is too low high e Always check the current at the beginning of the run The current may be too low or too high for a particular voltage setting If the buffer is prepared improperly the conductivity may be too low or too high and not enough power will be delivered to the cell S
31. tings The actual power supply reading may be lower throughout the run These conditions are excellent for neutral proteins pI 6 0 as we found at least 90 of the carbonic anydrous protein transferred successfully Table 3 4 DNA and RNA These conditions were determined empirically using 5 uniform TBE Criterion gels and the low range Fluorescein labeled DNA standards catalog number 170 3123 Buffer 1X TBE see section 3 3 Buffer formulation Criterion Blotter Overnight 12 hrs 30 minutes with plate electrodes 10 V 50 V 100 mA 750 950 mA Criterion Blotter Overnight 12 hrs 60 minutes with wire electrodes 20 V 50 V 100 mA 300 500 mA Note The power supply should be set on these maximum settings The actual power supply reading may be lower throughout the run 3 2 Notes on Electrophoretic Transfer Conditions 1 Pre equilibration of gels All gels should be pre equilibrated in transfer buffer prior to electrophoretic transfer may not be necessary for native gels and nucleic acid gels where transfer buffer is generally the same as running buffer Pre equilibration will facilitate the removal of contaminating electrophoresis buffer salts and neutralization salts If the 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 to various degrees depending on the acrylamide percentage in methanol buffers Equilibration allows th
32. tter cell plus another 300 to 500 ml for assembly procedure for a total of 1 6 to 1 8 liters of buffer It is efficient to use premixed buffer concentrates from Bio Rad where available Do not add acid or base to adjust pH of the following buffers Methanol should be analytical reagent grade because metallic contaminants in low grade methanol will plate on the electrodes Always add methanol ethanol last Note Some pH electrodes will not perform a proper measurement for the pH of Tris buffers If the pH of the buffer is off check to make sure the electrode is designed to work with Tris buffers If the pH electrode functions properly for Tris buffers and the pH is below 8 0 remake the buffer 1 SDS PAGE gels Towbin Buffer with 20 Methanol 25 mM Tris 192 mM glycine 20 v v methanol pH 8 3 a Use Premixed buffer concentrate solution 100 ml of 10X Tris Glycine buffer catalog number 161 0734 1L bottles or catalog number 161 0757 5L cube 700 ml of dd H O 200 ml of methanol b From dry reagents Mix 3 03 g Tris 14 4 g glycine in 600 ml of dd H O add distilled deionized water dd H O to 800 ml add 200 ml Methanol Towbin Buffer with 10 Methanol 25 mM Tris 192 mM glycine 10 v v methanol pH 8 3 a Use Premixed buffer concentrate solution 100 ml of 10X Tris Glycine buffer catalog number 161 0734 1L bottles or catalog number 161 0757 5L cube 800 ml of dd H O 100 ml of methanol b From dry reagents Mix 3 03 g Tr
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