HEP-1 and HEP-3 Semidry Electroblotter
Contents
1. eee Bey 3 quem om 8 Eum I SELL This list does not include all possible chemical incompatibilities and safe compounds Acrylic products should be cleaned with warm water a mild detergent such as Alconox and can also be exposed to a mild bleach solution 10 1 In addition RNAse removal products are also safe for acrylic 6 2 Semidry Electroblotter Thermo Scientific Section 7 Optional Equipment Contact Technical Services to order replacement parts Accessories 2245 Gu dod ord Catalog No Blotting Filter Paper 20cm x 20cm pkg of 100 FP 1 Blotting Filter Paper 35cm x 45 cm pkg of 100 FP 2 Blotting Filter Paper 46cm x 57 cm pkg of 100 FP 3 Blotting Filter Paper x pkg of 100 FP 4 Blotting Filter Paper 10cm x 10cm pkg of 100 FP 6 Blotting Filter Paper 12cm x 16cm pkg of 100 FP 7 Power Supply Leads icc ended PR em de PSL 5 Buffer Kit recommended for Western Blotting ER 35 ER 35 Electroblot Buffer Kit For optimal performance on Western blotting applications the ER 35 electroblot buffer kit is recommended These three different buffers anode 1 anode 2 and cathode 1 vary in buffering capacity and increase the transfer efficiency Thermo Scientific Semidry Electroblotter 74 E ERE SEI L006 OSI cL 6 0 98 uoneuuojul 10 10jnqujsip e90 Jno YSN au ep2. Catalog No Complete System e Lone a o HEP 1 Transfer Area eei e 20cm W x 20cm L Footprint 25cm W x 25cm D x 5cm H Complete System is 3 Transfer Area 2222 es 35cm W x 45cm L Footprint 40cm W x 51cm D x 5cm H Semidry Electroblotter 1 1 Section 1 General Information Power Supply Leads Heavy Duty Knobs x HEP 1 3 V HEP 3 4 Cathode Figure 1 1 Exploded View Table 1 1 Parts List base with stainless steel cathode e 1 lid with platinum titanium anode attached pair of power supply leads 3 knobs for the HEP 1 4 knobs for the HEP 3 1 2 Semidry Electroblotter Thermo Scientific Materials Needed Thermo Scientific Section 2 Setting Up Once the proteins or nucleic acids in a sample aliquot have been separated on a slab gel the resulting bands may be transferred to a solid support membrane The primary reason for this type of blot is one of localization and secondarily concentration of discrete bands Although many have used alternative cross linking agents such as DATD N N dihydroxyethylene bis Acrylamide to allow for the accessibility of gel bound proteins this still represents an impediment to radio enumeration due to the quenching by the gel matrix itself The most common solid support membrane is nitrocellulose A second type of membrane is PVDP Polyvinylidene difloride which is generally used when a
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5. 384 387 This paper discusses quantitative yields of proteins of different molecular weights using different transfer conditions Dunbar B S Ed 1994 Protein Blotting A Practical Approach IRL Press at Oxford University Press Oxford England A great guide to blotting techniques including visualization immunological techniques and sequence analysis Thermo Scientific Section 5 Technical Tips Semidry Blotting General Nucleic Acid Blotting References References cont 8 Trnovsky Jan Semidry Electroblotting of DNA and RNA from Agarose and Polyacrylamide Gels Bio Techniques 13 800 804 1992 9 Blotting Hybridization amp Detection An S amp S Laboratory Manual a publication of Schleicher and Schuell c 1995 This publication put out by a leading manufacturer of blotting membranes gives a good set of protocols for transfer of both proteins and nucleic acids 10 Hybond Blotting Guide The direct route to excellent blotting results Amersham Life Science This publication gives very helpful hints and tips for producing good Western Northern and Southern blots along with a useful reference list It also includes a very useful troubleshooting guide for nucleic acid and protein blots with pictures of the problems description of symptoms and proposed solutions Recipes for Buffers Tris Borate EOTA Buffer TBE 1X or 0 5X TBE is used for agarose gel electrophoresis and semidry elec troblotting o
6. Anderson J 1984 Electroblotting of multiple gels A simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide gels to nitrocellulose J Biochem Biophys Methods 10 203 209 This paper describes the semidry blotter with a 3 buffer system Owl ER35 which is effective for transfer of proteins Protein Blotting 3 Castora Frank 1 Western Blotting of Proteins Clinical Biotechnology 1 43 49 1989 This review article on Western Blotting gives a good overview of factors such as transfer buffers types of membranes and post membrane stains Although written for standard tank blotting much of this is applicable also to semidry blotting Eckerskorn Christoph and Lottspeich Friedrich Structural characterization of blotting membranes and the influence of membrane parameters for electroblotting and subsequent amino acid sequence analysis of proteins Electrophoresis 14 831 838 1993 A useful reference if you plan to do protein sequencing of samples transferred samples LeGendre Nancy Immobilon P Transfer Membrane Applications and Utility in Protein Biochemical Analysis BioTechniques suppl to vol 9 788 805 1990 This references deals specifically with transfer conditions using Immobilon P type membranes Tovey E R and B A Baldo 1987 Comparison of semidry and conventional tank buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes Electrophoresis 8
7. Mark the membrane to indicate the side to which the samples will be on This is important in the event that any successive probe is negative and to indicate sample orientation This can be done by either clipping a comer of the membrane or using a ball point pen Clip the same comer until you retire Wet the membrane according to its manufactures recommendations followed by a quick equilibration in transfer buffer It is often helpful to have all the filter paper and membranes sitting in transfer buffer as you start to build the blotting sandwich With this device the transfer is in the upward direction as shown in the figure below and the blotting sandwich is built upward upon the mirrored stainless steel plate of the bottom half of the blotter Lay three filter pads one after the other down Each should be soaking wet with buffer In fact the lower chamber may filled with buffer as you build the blotting sandwich Add a few mL of buffer to the filter pad and gently layer the gel Beginning at one end of the filter paper align the gel with the paper s edge and slowly lower the other end driving out any bubble Wearing gloves gently smooth out any bubbles by forcing them to the closest edge of the gel Test tubes and pipettes many also be used for this purpose Thermo Scientific Section 2 Setting Up Gel Sandwich Alternatively you can place the filter paper in a box with buffer toss in 1 the gel and drain th
8. of the counter top Slowly push the bottom plate back toward the counter top holding the top plate in place which will allow the blotting paper and gel to peel away from the top piece of glass The weight of the gel and blotting paper causes the gel to slowly peel away from the top plate Continue this process until the gel is almost entirely peeled away from the glass A small portion of the gel may still be attached to the glass in this case a small stream of water from a wash bottle can be used to aid in removal of the gel Semidry Electroblotter 2 3 Section 2 Setting Up 24 DNA Gels continued Gel Sandwich Assembly Semidry Electroblotter An alternative procedure at this step is to peel the paper with the gel attached from the glass plate upon which it rests Beginning at one end of the gel slowly lift and peel back the paper with the gel attached As before a stream of water from a wash bottle can be sprayed at any spot to aid separation of the gel from the glass plate Place the gel resting on the Whatman 3MM paper flat on a lab bench Wearing gloves cut a piece of nylon membrane the size of the gel and blotting paper Briefly but thoroughly wet the membrane in 0 5 x TBE in a shallow tray being careful not to crease the membrane Holding the membrane by two corners allow the excess liquid to drip from the membrane into the tray Wearing gloves cut the membrane to the size of the gel and blotting paper
9. the DIOE at sine oso de ge nia OR Ue 2 2 DNA GS 9520 es 2 3 Gel Sandwich Assembly race 2 4 Using the System 3 1 Running the Blot i103 eer eee hee eee ea 3 1 Transfer settings iio aA qe ei aan 3 1 Running Conditions so ores ge eee cod ae exa 3 2 Factors That Affect Transfer Efficiency ee e ep 3 2 Troubleshooting uso uiia eia exon ea n pr tn cena 6 4 1 Technical Tips ru REED 5 1 Semidry Blotting General References 5 2 Recipes for Dutti once eee hae eee ewes 5 3 Care and 0 6 1 Optional 7 1 Semidry Electroblotter V Thermo Scientific Section 1 General Information The Thermo Scientific semi dry electroblotter offers rapid transfer of proteins or nucleic acid molecules from polyacrylamide or agarose gels to membranes Solid plate platinum titanium and stainless steel electrodes are highly conductive and allow transfer at low voltages without external cooling systems Plate electrodes also provide a uniform electric field for efficient even transfers Before starting unpack the unit and inventory your order If any parts are missing contact Technical Services immediately You have 30 days from date of shipment to make any shipment claims Item Description
10. 2 hours Needs to experi 15 20 minutes at this 30 minutes to 2 hours Running Time mentally determined generally in the lower setting large molecules need range longer transfer times Thermo Scientific Section 4 Troubleshooting reme ar Transfer efficiency is poor e Current is too low Power supply is inappropriate for semidry transfer e Semidry transfer should be performed at constant current Current density should be between 0 5 and 3mA cm2 of stack surface area e Many power supplies will shut off or blow a fuse when run at the conditions required for semidry transfer Semidry transfer requires low voltage often less than 10V and high current Check with the man ufacturer of the power supply to determine whether it is appropriate for semidry transfer e Transfer performed for too short a time Increase the amount of time for transfer e Transfer sandwich was assembled in the wrong order oo much methanol in the transfer buffer High percentage gels restrict transfer e Puddles of buffer were present on the cathode allowing the current to bypass the stack e The filter paper was too dry e The pH of the transfer buffer is too close to the isoelectric point of the protein Thermo Scientific The semi dry electroblotter is configured with the cathode on the bottom and anode on top This means that an upward transfer is being performed rather than downward Follow the instructions ca
11. Semidry Electroblotter Models HEP 1 and HEP 3 Operating and Maintenance Manual 7007332 Rev 0 Visit us online to register your warranty www thermoscientific com warranty 69 Preface MANUAL NUMBER 7007332 0 5 1 12 Transfer to Marietta was The Panther 3 2003 CCS REV ECR ECN DATE DESCRIPTION By Thermo Scientific Semidry Electroblotter i Preface CAUTION Contains Parts and Assemblies Susceptible to Damage by Electrostatic Discharge ESD Important Read this instruction manual Failure to read understand and follow the instructions in this manual may result in damage to the unit injury to operating personnel and poor equipment performance A Caution All internal adjustments and maintenance must be performed by qualified service personnel A Warning To avoid the risk of personal shock always disconnect the gel box from the power supply Further the power supply must be equipped with a shut down on disconnect circuit Running conditions for this unit should not exceed the name plate readings found on the lower buffer chamber A This system is designed to meet IEC 1010 1 safety standards IEC 1010 1 is an internationally accepted electrical standard for laboratory instruments Statement of Proper Use Use this product only for its intended purpose as described in this manual Do not use this product if the power leads are damaged or if any of its surfaces are cracked Material in this manual is
12. e box until the gel falls to the pad Add few mL Assembly continued of buffer to the filter pads already in the bottom chamber Then pick up the paper and gel and layer them on the filter stack 9 Adda few mL of buffer to the gel and gently layer the membrane as you did the gel 10 Repeat with three more pieces of filter paper 11 Holding the stack drain off all excess buffer from the plate Wipe away any droplets around the edge of the stack Anode Filter Paper Transfer Membrane Gel E E Filter E Paper Cathode Figure 2 1 Assembly Order Thermo Scientific Semidry Electroblotter 2 5 Running the Blot Thermo Scientific Transfer settings Section 3 Using the System Place and loosely tighten down the lid with the supplied black knobs The weight of the lid is usually enough and the screws are not required Note Do Not Use the screws for gels thicker than 1 5mm or when using more than 6 filter pads total If the screws are used tip the unit on an angle to drain off any excess buffer that may have squeezed out This will safely remain in the moat around the electrode plate 2 Attach the power leads red to red black to black to an appropriate power supply The red lead has a shroud that will stop it from attaching to the black cathode 3 Run the blot Blott
13. e ethanol or other organic solvents to clean these products Do not autoclave bake or microwave your unit Temperatures over 50 C can damage acrylic A Note If an RNase free electrophoresis system is desired there are various methods to rid the system of RNA contamination For fast and easy decontamination use RNase AWAY Spray wipe or soak labware with RNase Away then wipe or rinse the surface clean it instantly eliminates RNase RNase Away eliminates the old methods that include treatment with 0 1 96 Diethyl Pyrocarbonate DEPC treated water and soaking in diluted bleach DEPC is suspected to be a carcinogen and should be handled with care This electrophoresis system should never be autoclaved baked or placed in a microwave A To order RNase Away contact Technical Services P N 21 236 21 250ml bottle P N 21 402 178 475ml spray bottle P N 14 375 35 1 liter bottle P N 14 754 34 4 liter bottle Rnase AWAY is a registered trademark of Molecular BioProducts Inc Semidry Electroblotter 6 1 Section 6 Care and Cleaning Table 6 1 Chemical Compatibility for Acrylic Based Products erem ___5 CO Mew mersa Tamm EA e eem p opem CI IS CC CECI cion rar RN Bee n DN Bem CO CO Hydrogen peroxide 28 solution ______ e e E DN ETC CN pememem O Deme qr
14. e glass plates where the spacer had been present Gently rock the spatula forcing separation of the plate from the gel The gel will normally remain affixed to the bottom not notched plate Remove the top notched plate by slowly lifting it from the side with the inserted spatula and gradually increasing the angle Imtil the top plate is completely separated form the gel If the gel sticks to the top plate in an isolated spot a stream of water from a wash bottle can be sprayed at the spot to aid separation Occasionally a gel sticks to the top plate instead of the bottom plate In this case flip the gel sandwich over and follow the same procedure Once the plates are separated remove the second side spacer along with any extraneous bits of acrylamide around the gel If excess water was utilized to aid in the separation of the gel from the glass plate use a paper towel to absorb the excess liquid Using a piece of dry blotting paper Whatman 3MM chromatography paper 46 x 57cm gently roll it onto the gel beginning at one end and working slowly towards the other end Care should be taken to prevent air bubbles from forming between the paper and the gel Once the blotting paper is in position set another glass plate on top of the paper Flip the entire sandwich over The gel should now be resting on top of the filter paper between the two pieces of glass Position the sandwich with one long side of the glass hanging about one third of the way off
15. e in transfer buffer e Membrane was dried out before it was added to the transfer sand e Alcohol was not used to prewet e PVDF is hydrophobic and requires a short soak the membrane in methanol prior to transfer Roll a test tube or pipet over the membrane e Air spaces are interfering with make sure it is clean before putting the rest of contact between the gel and the the filter paper on the sandwich Transfer will not membrane occur where the gel is not in contact with the membrane e Running conditions sample preparation per e Electrophoretic conditions were centage acrylamide and many other variables incorrect or not ideal can affect the migration and resolution of pro teins Review your electrophoresis conditions e Transferring at too high a current e Refer to the Running Conditions table Always pre wet the membrane according to the manufacturer s instructions White spots indicate the dry areas of the membrane Membrane was not thoroughly e Running at constant voltage can cause power fluctuations that will cause overheating A buffer that has not been made correctly or that has too high in ionic strength can also burn a gel by over heating A cracked and dry gel often is an indica tor of overheating oo much current Thermo Scientific Thermo Scientific Section 5 Technical Tips How long will it take to blot the proteins from my gel Transfer times have to be determined experime
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17. f nucleic acids Final 1X composition 89mM Tris Base 89mM Boric Acid 2mM disodium EDTA pH 8 3 1X Towbin Buffer 1X Tris glycine buffer Towbin buffer minus the methanol is used for agarose and polyacrylamide gel electrophoresis of nucleic acids Towbin buffer containing 20 methanol is a commonly used buffer for semi dry transfers 0 025M Tris Base 0 192M Glycine 20 MeOH pH 8 3 Thermo Scientific Semidry Electroblotter 5 3 Section 5 Technical Tips Recipes for Buffers 1x Tris Borate EOTA Buffer TAE continued 1 X TBE is used for agarose and polyacrylamide gel electrophoresis and semidry electroblotting of nucleic acids Final 1 X composition 0 04M Tris Acetate 0 001 M disodium EDTA pH 8 0 1X Tris Glycine SOS Buffer TGS 1 X TGS buffer is used for denaturing polyacrylamide gel electrophoresis of proteins Final 1 X composition 0 025M Tris Base 0 192M Glycine 0 1 SDS pH 8 3 1X Three Buffer System for Semidry Electroblotting This buffer is used with the HEP 1 Semidry Electroblotter Final 1 X composition Anode 1 Buffer 0 3M Tris Base 20 MeOH pH 10 4 Anode 2 Buffer 0 025M Tris Base 20 MeOH pH 10 4 Cathode Buffer 0 025M Tris Base 0 04M Caproic Acid 20 MeOH pH 9 4 NAQ Northern Transfer Buffer 8 For transfer of RNA from agarose gels With its high buffering capacity and low ionic strength this buffer is more efficient than TAE TBE or MOPS from agarose gels 50X 0 2M morpholi
18. for information purposes only The contents and the product it describes are subject to change without notice Thermo Fisher Scientific makes no representations or warranties with respect to this manual In no event shall Thermo Fisher Scientific be held liable for any damages direct or incidental arising out of or related to the use of this manual 2012 Thermo Fisher Scientific All rights reserved Semidry Electroblotter Thermo Scientific Preface Important operating and or maintenance instructions Read the accompanying text carefully gt gt S Potential electrical hazards Only qualified persons should perform procedures associated with this symbol Su 9 Pi Equipment being maintained or serviced must be turned off and locked off to prevent possible injury Hot surface s present which may cause burns to unprotected skin or to materials which may be damaged by elevated temperatures Marking of electrical and electronic equipment which applies to electrical and electronic equipment falling under the Directive 2002 96 EC WEEE and the equipment that has been put on the market after 13 August 2005 This product is required to comply with the European Union s Waste Electrical amp Electronic Equipment WEEE Directive 2002 96 EC It is marked with the WEEE symbol Thermo Fisher Scientific has contracted with one or more recycling disposal companies in each EU Member State European Country and this product sh
19. ing takes place at a given migration rate for a specified time The units are mA times hrs If you need to slow the transfer down to say coincide with the setting up of a probe simply decrease the current mA to match the added time you require mA hr Std setting mA hr New setting Alternatively the current can be increased to decrease the time This assumes that you have determined an initial mAh value that works well for the molecules you are interested in A current to use for a 45 min time period is based on the area cm cm of your gel which is the resistor in this system A range of 0 8 to 2 mA per square centimeter of gel For example if you had a 10 x 10cm gel the area would be 100cm so the current range would be 80mA 0 8mAcm x 100cm to 200 mA 2mAcm x 100cm Blots may also be run at constant voltage Some power supplies have difficulty sustaining steady voltages at these low voltage settings If you find that voltages are fluctuating or that the power supply shuts itself off when set on constant voltage use constant current settings instead Semidry Electroblotter 3 1 Section 3 Using the System 3 2 Transfer Settings continued Factors That Affect Transfer Efficiency Running Conditions Semidry Electroblotter Read your power supply s instructions to ensure that the power supply will work at a voltage lower than IOV These voltages often occur in semidry blotting Contact the manufac
20. isino eio eds pue sonas uonejedo AjueJeM jueuudinbe uo suonsenb noA Jamsue peaJ 9J 9M lt 9 0 1 Jo epeue pue VSN 158y 8 y 008 1 1e jueuniedeq samas 1no p nb s eor es y eAnueAeJd pue uonejejsur 3ueuudinbo AjjnJoued uononujsur jueuudinbe 910 99 uoneuuojJur uoneJedoud djay peaiJ si sejes INOA SsjonpoJd Jo sso JO S oJd 1so uonejyuui jenuenbesuoo Jo apu Aue eq jou eys ouueu AlddV TIVHS 3ISOdYINdA YVINOILYVd V SSANLIS HO ALETISVENVHOHZSIN JO S3LLNVSHSHVM ON Cal dW YO N3 LLIHM YSHLAHM S3LLNVHHVM H3HLO 11 30 NA NI ANY SAISNIOXS SI ALNVHSHVM SIHL uoneunsep go4J peddius sued pue pied 0 peuJunjaJ eq 1snuu sued BuiuuJojuoo uou uondo s ouueu 3ueuudinbe Jo jueuoduuoo Aue JO Jsnw seorues e2iuuo9 99 AueJew jeuiBuo puo Keq y o JO jueuudinbe ay JOYS jueJem y JOU uenem siu Jepun juauwdinba Jo sued 1ueuoduJoo Jo Jeda JO
21. nopropanesulfonic acid MOPS 50mM sodium acetate 5mM EDTA pH 7 0 5 4 Semidry Electroblotter Thermo Scientific Recipes for Buffers Thermo Scientific continued Section 5 Technical Tips NAQ Southern Transfer Transfer of DNA from agarose gels 50X 1M ethanolamine glycine buffer pH 11 NAQ Transfer Buffer 10X 0 8M Tris 1 18M borate 24mM EDTA pH 8 3 CAPS Buffer pH 11 This buffer can be used to improve transfer of some proteins 10mm CAPS 3 cyclohexylamino 1 1 0 methanol propanesulfuric acid adjust to pH 11 with sodium hydroxide Semidry Electroblotter 5 5 Thermo Scientific Section6 Care and Cleaning Care of Acrylic The following chemical compatibility chart is supplied for the convenience of our customers Although acrylic is compatible with most solvents and solutions found in the biochemical laboratory some solvents can cause substantial damage Keep this chart handy to avoid hann to your apparatus by the use of an inappropriate solvent Codes S Safe No effect except possibly some staining A Attacked Slight attack by or absorption of the liquid Slight crazing or swelling but acrylic has retained most of its strength U Unsatisfactory Softened swollen slowly dissolved D Dissolved In seven days or less A Few Tips About Caring for Your System Warning Organic solvents cause acrylic to craze or crack Clean all acrylic systems with warm water and a mild detergent Do not us
22. ntally This is because transfer time is dependent upon Percentage of gel Type and amount of cross linking in the gel Type of protein cytoplasmic membrane nuclear Size of protein There is no formula for determining transfer time There are too many variables involved to give specific transfer conditions that will work for every protocol Guidelines are e 2mA cm2 of gel for 1 hour These guidelines are just a starting point and exact conditions have to be determined Different kinds of blotting Western Blotting is a blotting method for proteins that use specific antibodies attached to particular protein to help identify it It is often performed after SDS PAGE or some other form of polyacrylamide gel electrophoresis Southern Blotting is a method sometiems called hybridization because a radioactive probe is hybridized or attached to specific pieces of DNA Northern Blotting is a similar method but the molecules involved are RNA Both Southern and Northern blotting generally require the DNA or RNA to first be separated out on an agarose gel Semidry Electroblotter 5 1 Section 5 Technical Tips 5 2 Semidry Blotting General References Semidry Electroblotter me Bjerrum O J and Schafer Nielsen C in Dunn J J ed Electrophoresis 86 VCH Weinheim 1986 pp 315 327 These authors compare results using different transfer buffers Towbin buffer vs the three buffer system Khyse
23. of your power supply to determine if it will work for tank blotting applications Contact Technical Services for power supply recommendations if you do not have an appropriate power supply Semidry Electroblotter 2 1 Section 2 Setting Up 2 2 Materials Needed continued Setting Up the Blot Semidry Electroblotter Blotting buffer The most commonly used buffer for protein blotting from polyacrylamide gels is Towbin buffer Small amounts of buffer may be needed for equilibrating the gel and membrane prior to blotting in addition to the buffer in the transfer tank Should be cooled to 40 C Filter paper Sometimes called blotting paper it used in the blotting sandwich Blotting membrane Nitrocellulose and PVDF Polyvinylidene difloride can be used for proteins while charged Nylon membranes can be used for nucleic acids The choice depends upon the user s preference and sometimes the detection method to be used There are three types of gels each requiring a different handling procedure before they can be added to the blotting sandwich Protein and agarose gels set up are arranged together with sequencing gel transfer protocol following separately DNA RNA If these gels were not run in IXTBE they should be equilibrated for 10 minutes in this buffer Protein Gels After electrophoresis remove the gel assembly from the apparatus and remove the spacers Open the gel cassette by gently rocking a spatula between
24. ould be disposed of or recycled through them Further information on Thermo Fisher Scientifics compliance with this directive the recyclers in your country and information on these products will be available at www thermofisher com Y Always use the proper protective equipment clothing gloves goggles etc Y Always dissipate extreme cold or heat and wear protective clothing Y Always follow good hygiene practices Y Each individual is responsible for his or her own safety Thermo Scientific Semidry Electroblotter iii Preface Do You Need Information or Assistance on Thermo Scientific Products If you do please contact us 8 00 a m to 6 00 p m Eastern Time at 1 740 373 4763 Direct 1 800 438 4851 Toll Free U S and Canada 1 877 213 8051 FAX http www thermoscientific com Internet Worldwide Web Home Page service led marietta thermofisher com Tech Support Email Address www unitylabservices com Certified Service Web Page Our Sales Support staff can provide information on pricing and give you quotations We can take your order and provide delivery information on major equipment items or make arrangements to have your local sales representative contact you Our products are listed on the Internet and we can be contacted through our Internet home page Our Service Support staff can supply technical information about proper setup operation or troubleshooting of your equipment We can fill your needs for spare or replacement parts o
25. r provide you with on site service We can also provide you with a quotation on our Extended Warranty for your Thermo Scientific products Whatever Thermo Scientific products you need or use we will be happy to discuss your applications If you are experiencing technical problems working together we will help you locate the problem and chances are correct it yourself over the telephone without a service call When more extensive service is necessary we will assist you with direct factory trained technicians or a qualified service organization for on the spot repair If your service need is covered by the warranty we will arrange for the unit to be repaired at our expense and to your satisfaction Regardless of your needs our professional telephone technicians are available to assist you Monday through Friday from 8 00 a m to 6 00 p m Eastern Time Please contact us by telephone or fax If you wish to write our mailing address is Thermo Fisher Scientific 401 Millcreek Road Box 649 Marietta OH 45750 International customers please contact your local Thermo Scientific distributor iv Semidry Electroblotter Thermo Scientific Thermo Scientific Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Table of Contents General Information 1 1 Setting UD nose des PEE 2 1 Materials Needed ass den x SERE CE ER oaths tee 2 1 Setting Up
26. re fully when assembling the transfer sandwich Try a more acidic or basic transfer buffer e Reducing methanol can help elute proteins from the gel but can reduce binding to nitrocellulose membranes e Higher percentage acrylamide or crosslinker can restrict elution of proteins Use the lowest percent age acrylamide possible to separate your proteins e Always clean up the lower plate before closing the lid of the transfer apparatus Do not squeeze the stack excessively as this also creates puddles that the current can pass through e Filter paper should be saturated with transfer buffer before adding them to the sandwich Semidry Electroblotter 4 1 Section 4 Troubleshooting 4 2 Semidry Electroblotter Problem Nitrocellulose membranes Insufficient binding of proteins to the mem Smeared or swirled transfer and missing Use 0 2micron pore size nitrocellulose instead of 0 45micron or use PVOF with a higher binding capacity Over transfer through the membrane Not enough methanol in the e Nitrocellulose binds proteins best when 20 transfer buffer methanol is used in the transfer buffer e Low MW proteins are not Use glutaraldehyde to crosslink the proteins to binding well or are being washed the membrane and use Tween 20 in the wash away steps e Membrane should be completely gray and slightly translucent when added to the sandwich If it has dried out rewet in methanol and equili brat
27. the plate forcing separation of the plate from the gel The gel will normally remain affixed to the bottom plate Remove the top notched plate by slowly lifting it from the side with the inserted spatula and gradually increasing the angle until the plate is completely separated from the gel If the gel sticks to the top plate in an isolated spot a stream of water from a squirt bottle can be sprayed at the spot to aid separation Remove the gel from the remaining plate Tip the plate up side down and start one edge and allow it to roll off into transfer buffer Alternatively place the plate with the gel attached into transfer buffer Incubate the gel in transfer buffer for 15 min with gentle agitation If the gel is on the plate it will become loose during this step Thermo Scientific Thermo Scientific Section 2 Setting Up DNA Gels Preparing a sequencing gel for transfer on the HEP 3 l Remove the gel cassette from the gel box and place under cold running tap water until the surfaces of both glass plates are cool This facilitates handling of the gel and prevents the gel from curling as it cools once it is removed from between the glass plates Place the glass plate sandwich with the notched or shorter plate facing up flat on paper towels on the lab bench and remove any excess liquid and remaining tape or binder clips Remove one side spacer wearing protective gloves and insert a long metal spatula between th
28. transferred protein is to be sequenced additionally it has a 2x binding capacity Also used for nucleic acid capture are Nylon membranes In either case the proteins are transferred from the gel to the matrix in an electric field perpendicular to the gel initial running direction Tris based buffers are employed in the transfer Methanol and SDS are modifiers often use in protein transfer buffer These components however are antithetical in their effects both in terms of movement and adsorption Methanol restricts protein movement from the gel but is often required to support the ionic nature of protein to nitrocellulose binding SDS aids in protein elution but can also inhibit binding of small molecular weight proteins Mozdzanowski J High yield electroblotting Electrophoresis 1992 Vol 13 p 59 64 In order to use this blotting device you will need Power supply Blotting requires a power supply that can operate at a fairly high current setting and low voltage If an inappropriate power supply is used the power supply may blow a fuse shut itself off display a no load or short load message or even have a short circuit It is very important to be sure that the power supply you will be using will work with this device Some power supplies that will work with this device are Catalog No OSP 135 OSP 300 EC Apparatus EC135 and EC570 and Bio Rad s PowerPac 200 If your power supply is not among those listed contact the manufacturer
29. turer regarding the unit s performance under high current low voltage conditions if you have any questions While general conditions can be described which will result in successful transfer of most molecules it should be noted that optimal transfer conditions will vary based on the characteristics of the molecule you are working with Some factors that affect transfer rate and efficiency include molecule size charge gel thickness and percentage and hydrophobicity The reference list at the end of this manual provides useful information that can help you choose optimal conditions for efficient transfer of a specific molecule HEP 3 Sequencing gel see HEP 1 set Protein tings for other size DNA RNA gels on this unit FP 1 20 x 20cm FP 6 Filter Paner 9 x 9cm FP 4 10 x FP 2 35 x 45cm or FP 1 FP 4 FP 5 FP 6 or P 10cm FP 7 12 16cm FP 3 46 x 57cm FP 7 or FP 5 18 x 20cm Nitrocellulose 45 or Membrane 0 2u PVDF 0 45p or 0 2u Towbin Buffer Buffer Electroblot Buffer Kit Transfer Buffer ER 35 3 Buffer System 0 5X 1X TBE 0 5X 1XTBE TAE Bjerrum and Schafer Nielsen Power Supply OSP 135 or equivalent SION cen Ow volt OSP 135 or equivalent age power supply Constant current 0 8 For entire gel 1200 Constant Current 0 5 3mA per cm2 gel sur 1400 mA approximate 3mA cm2 gel surface Rowen SEMIS face area 10 14 Volts ly 0 8mA cm2 area 10 14 Volts maxi maximum produces 5 8V mum 30 minutes to
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