Fastblot user manual (English)
Contents
1. 10 Acetic acid 45 bidest water Slow destaining in 25 Isopropanol 10 Glacial acid 65 bidest water Final destaining in 7 Acetic acid in bidest water Fastblot Instruction Manual 2009 07 21 biometra a part of Analytik Jena 8 Trouble Shooting Corrective Actions U Wrong order of membrane and Reverse order of membrane gel and gel gel on top of membrane Wrong connection to the Power Cexchange connection Supply Red anode Black cathode Acidic buufer system transfer Place transfer membranes on towards cathode cathode side lid and gel on anode side base Incomplete transfer Transfer time too short check by Increase trander time gel staining Tranfer time too long blow Reduce transfer time Use through i e proteins are nitrocellulose with smaller stained on both sides of the pore size or PVDF membrane mebrane Irregular transfer Air bubbles in the blot sandwich Remove air bubbles by gently roling e g a pasteur pipette over the emebrane Generation of air bubbles with Reduce transfer time chill long transfer times by buffer before use electrolysis and heating up Irregular current flow as size of Transfer membranes and blotting papers and transfer blotting papers must be cut emebrane are large than gel size exactly to gel size membrane prewetting membrane Proteins are stained on Transfer time to long blow Reduce transfer time Use both sides of the throug2. Senders name and address b Name of a contact person for further inquiries with telephone number C Precise description of the fault which also reveals during which procedures the fault occurred if possible 26 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 13 Equipment Decontamination Certificate To enable us to comply with german law i e 871 StrlSchV 817 GefStoffV and S19 ChemG and to avoid exposure to hazardous materials during handling or repair please complete this form prior to the equipment leaving your laboratory COMPANY INSTITUTE ADDRESS PHONE NO FAX NO E MAIL EQUIPMENT Model Serial No If on loan evaluation Start Date Finish Date Hazardous materials used with this equipment Method of cleaning decontamination The equipment has been cleaned and decontaminated NAME POSITION HEAD OF DIV DEP INSTITUTE COMPANY SIGNED DATE PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRA DISTRIBUTOR TOGETHER WITH THE EQUIPMENT PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING INSTRUMENTS WITHOUT THIS CERTIFICATE ATTACHED WILL BE RETURNED TO SENDER Fastblot Instruction Manual 2009 07 27 biometra a part of Analytik Jena General Information for Decontamination Please contact your responsible health amp safety officer for details Use of radioactive substances Please contact your responsible person for details Use of genetically change organis
3. e Return only defective devices For technical problems which are not definitively recognisable as device faults please contact the Technical Service Department at Biometra Tel 49 0 5 51 50 88 1 10 or 12 Fax 49 0 5 51 50 88 1 11 e mail Service biometra com e Please contact our service department for providing a return authorization number RAN This number has to be applied clearly visible to the outer box Returns without the RAN will be not be accepted e Important Carefully clean all parts of the instrument from residues and of biologically dangerous chemical or radioactive contaminants lf an instrument is contaminated Biometra will be forced to refuse to accept the device The sender of the repair order will be held liable for possible damages and losses resulting from insufficient decontamination of the device e Please prepare written confirmation use the Equipment Decontamination Declaration following on the next page that the device is free of biologically dangerous chemical or radioactive contaminants This confirmation must be attached to the outside of the packaging e Use the original packing or a similarly robust packing when returning the device If not available contact Biometra or your local distributor e Label the outside of the box with CAUTION SENSITIVE INSTRUMENT and the RAN number sticker Attach the Decontamination Declarartion e Please enclose a note which contains the following a
4. Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 1 3 Legal Notes Copyright All rights reserved It is not allowed to copy and publish the manual or parts of it in any form as copies micro film or other methods without a written authorisation from Biometra Biometra is pointing out that applied company and brand names are usually protected trade marks Liability Biometra is not liable for damages and injuries caused by use not considering these operatin instructions in parts or completely 1 4 Meaning of the Instructions Biometra recommends that you first read these instructions carefully Then assemble and disassemble the apparatus to become familiar with the system After these preliminary steps you should be ready to transfer a sample This operaton instruction is part of the product and should be kept over the full life time of the instrument It should also be forwarded to subsequent owners and users Make sure that additions and updates are inserted into the operation instructions Fastblot Instruction Manual 2009 07 5 biometra a part of Analytik Jena 2 Safety and Warning Notices 2 1 Definition of Symbols Symbol Definition Caution Refer to instruction manual Danger High voltage Fragile Using with direct current DC ipbPb N N General Safety Instructions A Delicate instuments Handle with care Do not operate this equipment if buffer or water leaks from the instr
5. naeh enter ads 9 31 DEODEOL SUV IG Pe m 9 3 2 Unpack and Check anne nern en en rer 9 3 3 Installation Conditions 022002220020000nnnennnennnnnnnnnnnnnnnnnennnnennnenennnnnnenenen nennen 9 3 4 Connecting CondaltlOrs oani oorr iaa rio inp Un ann 9 4 Operating Elements Controls 2202020002n000000nan0nannnnnnnnnnnun nun nun nnnnnnnnun nennen 10 3 OAM niea e E Ei E EEE 11 Sl PIOLEIN BIONNG eeaeee E ee een 11 5 1 1 Preparing a Blotting Sandwich ueeeseeeseeesseeeeeeereennn ne 11 5 1 2 Using a Discontinuous Buffer System sseesseessesssessseeeeeeenen nenne 14 5 2 DNA Blotting Agarose Gels sessessssssssssseseeneenn enean nnn nnn nns 16 5 2 1 Sample Preparation ccccccccsecceeceeeeeeeeceeeceecceeceneeeneeeueeeseeeetseetseeeeeeaeeeaseeaaes 16 5 3 Stacked Gel transfer EE ee 16 6 General Blotting Information 22222020200200000000 n0nannonnnunannnnnnnnnnnun nun nun nun nnnnn 17 6 1 Efficiency Of INE Transfer unse nie ee 17 6 2 Nitrocellolose Blotting Membranes 22202220200000000000n0n0n0nnnn ann nnne nnn 17 ON POUNG COIN ol RECIENTES 17 04 ERG E E 18 7 Buffers and Staining Destaining Solutions eeeeeeee eere nene 19 7 1 Continuous Buffer Systems sessssesse
6. or altered in any way Alteration of this instruments will void the warranty void the EN61010 1 certification and create a potential safety hazard Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 3 Setting Up Operation 3 1 Scope of Delivery The Fastblot comes complete with Body with electrode anode B33 B43 including cooling device Lid with electrode cathode Wires permanently attached to the lid with safety connectors Warranty Registration card Manual 3 2 Unpack and Check Unpack and carefully examine the Fastblot Report any damage to BIOMETRA Do not attempt to operate this device if physical damage is present Please keep the original packing material for return shipment in case of service issues A I Attention A Please fill out and send back the warranty registration card This is important for you to claim full warranty 3 3 Installation Conditions Place the chamber in proximity to the Power Pack with which it is to be connected Be sure to place the chamber in a safe dry location away from the edge of the working surface 3 4 Connecting Conditions The Fastblot has been designed to operate with D C current it Warning The Fastblot must not be earthed Fastblot Instruction Manual 2009 07 9 biometra a part of Analytik Jena 4 Operating Elements Controls Stainless steel cathode Connection for flow through cooling reflected in cathode Safe
7. shown in Fig 2 filter paper SEE EI m Fig 2 SS 000 seseeyenkege LUN LA oos NET blotting paper gel and Me nitrocellulose membrane blotting membrane 3 between the plate WADA SHRI ai electrodes Blotting paper 3 and blotting membrane are olate electrode anode eRe RN mE eer used in the same size as the gel Step 10 Switch on your Power Supply e g Biometra Standard Power Pack P25 Code No 040 800 or 048 850 or one of the Biometra High Voltage Power Packs at a constant current of 0 8 1 0 mA per cm of gel max 3 5 mA per cm of gel B64 resp max 5 mA per cm of gel Typical blotting times are approx 30 min Smaller molecules might be transferred within 5 min depending on the charge 8 Watts W resp 10 W B64 The electrodes consist of different materials DO NOT change polarity of the electrodes The anode is on the body of the Fastblot and the cathode is N Attention The maximum power for the B33 B34 B43 and B44 is A fixed on the lid 12 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena Step 11 If the lid is getting warm during the blot procedure reduce the current A The maximum temperature must not exceed 50 C Step 12 If you are using the water cooling please note the following The connection for the water cooling with the smaller diameter is the inlet the connection with the bigger diameter is the outlet Attention Never mix up
8. 3 B44 can be cleaned with a mild abrasive to remove salt that may deposit during normal operation The entire unit can also be periodically disassembled and cleaned with warm water to remove salt deposits Do not use alcohol gt 10 e g methanol ethanol or organic solvents e g acetone for cleaning of electrodes or housing The system should never be autoclaved or placed in a microwave 11 2 Servicing Regular servicing is not necessary Nevertheless cleaning of the electrodes should be done using a damp cloth wetted with dest water after each transfer In case that the electrodes are clogged after longer time of use and the blotting transfer is inhomogeneous or reduced please contact the Biometra Service Department 11 3 Replacement of Spare Parts Only original spare parts mentioned in these operating instructions are allowed 11 4 Other Accessories There are no accessory parts for this instruments Fastblot Instruction Manual 2009 07 25 biometra a part of Analytik Jena 12 Service Should you have any problems with this unit please contact our service department or your local Biometra dealer Biometra GmbH Service Department Rudolf Wissell Stra e 14 16 D 37079 Goettingen Phone 49 0 5 51 50 68 6 10 or 12 Fax 49 0 5 51 50686 11 e mail Service biometra com If you would like to send the unit back to us please read the following return instructions 12 1 Instructions for return shipment
9. be performed in the Fastblot B64 and up to four layer transfer can be performed in the Fastblot B33 B34 B43 and B44 The use of dialysis membranes between the layers is recommended may not be neccessary Prepare gels membranes and blotting paper as described above using continuous buffer systems When setting up the transfer stack place pre wetted blotting paper on top of the first gel followed by the second membrane second gel paper and then the cathode lid Measure the surface area of the top of the stack to determine proper current 16 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 6 General Blotting Information 6 1 Efficiency of the Transfer The efficiency of the transfer can be checked by applying Coomassie blue pre stained standard proteins on the gel After the blotting it is possible to stain the gel with Coomassie blue to check the completeness of transfer 6 2 Nitrocellolose Blotting Membranes High Quality Nitrocellulose membranes are standard for protein and nucleic acid blotting They can easily be stained for total protein by a dye stain e g Amido Black Coomassieblue Ponceau S Fast Green etc Nitrocellulose has a high binding capacity of apprx 80 90 ug cm Unspecific protein binding sites are easily and rapidly blocked avoiding subsequent background problems omall proteins esp 20 000 daltons may be lost during washing steps after the transfer Use of a smal
10. biometra a part of Analytik Jena Fastblot Semi Dry Electrophoretic Transfer Apparatus Instruction Manual Order No 014 100 014 200 015 100 015 200 015 600 I Warning Please read these instructions carefully before using this apparatus Biometra GmbH Service Department Rudolf Wissell Str 30 Rudolf Wissell Str 14 16 D 37079 Goettingen D 37079 Goettingen Tel 49 0 5 51 50 68 6 0 Tel 49 0 5 51 50 68 6 10 or 12 Fax 49 0 5 51 50 68 6 66 Fax 49 0 5 51 50 68 6 11 e mail Info biometra com e mail Service biometra com internet http www biometra de biometra a part of Analytik Jena This document describes the state at the time of publishing It needs not necessarily agree with future versions Subject to change Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena Contents 1 AUFOGUC UO I sorainak EEEE OEE EEEN 3 1 1 FIEIS OTD OCA OM et mE 3 1 2 SO SCHIC AT ONS zumeist een een near nenne Teen TEN ER 4 1 3 Legal NOGS MT 5 1 4 Meaning of the Instructions eeessessssssesssseseennnneneneeneennne ener nnns 5 2 Safety and Warning Notices uus 2200200002n0nannonnnnnnnunnnnun nun nnnnnnnn nun nnnnnnnnnnnnnnunnnn nenn 6 2 1 DEINEN OL VINO IS eure ee ee ee 6 2 2 General Safety Instructions u02200224002000nnnnonnnenennonnnnnnnnn anne nenn nano nenne nnns nnns 6 3 Sset ng UD Operation
11. de buffer for five minutes Step 2 Moisten the anode with anode buffer l Step 3 Cut blotting paper to the size of the gel Step 4 ooak blotting paper in anode buffer and place them on the anode Step 5 A further layer of the adapted blotting paper is soaked with the anode buffer II and added to the sandwich otep 6 Pre wet the membrane Nitrocellulose cut to the size of the gel with anode buffer Il PVDF membranes have to be successively incubated in methanol 1 2 seconds in H2O 5 minutes and in anode buffer Il Subsequently the membrane is placed on the transfer sandwich Step 7 The SDS Polyacrylamide gel is put on top of the membrane Step 8 Three layers of blotting paper are soaked in cathode buffer and placed on top of the gel Note Avoid the inclusion of air bubbles in the blotting sandwich and wear gloves Step 9 Connect the lid with the body of the Fastblot Take care that lid and body are attached completely horizontally Step 10 If you have assembled the sandwich for blotting the set up should look as shown in Fig 3 14 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena BE u Cathode Buffer Gel M PVDF Membrane Anode Buffer II Anode Buffer I Fig 3 Set up of filter paper blotting paper gel and blotting membrane between the plate electrodes Blotting paper and blotting me
12. e in two different sizes 16 cm x 20 cm and 23 5 cm x 28 5 cm electrode surface The size 16 cm x 20 cm is available with or without internal cooling The large size includes a paasive cooling option Warming up of the apparatus and blot sandwich during the transfer at high current conditions or during blotting of native proteins can be compensated by the internal cooling system of the Fastblot B33 or B43 B43 B44 B33 B34 B64 Fastblot Instruction Manual 2009 07 3 biometra a part of Analytik Jena 1 2 Specifications Construction Fastblot body PMMA Anode B33 B34 B64 Plasticised carbon B43 B44 Platinum coated titanium Cathode B33 B34 B64 Plasticised carbon B43 B44 Stainless steel Cooling option B33 B43 flow through Overall size L x W xH B33 B43 26 cm x 22 cm x 11 cm B34 B44 24 cm x 22 cm x 11 cm B64 34 cm x 44 cm x 14 cm Weight B33 B34 2kg B43 B44 2 5 kg B 64 6 kg Maximum gel size B33 B34 B43 B44 15 5 cm x 19 5 cm B64 23 cm x 38 cm Chemical compatibility The semi dry blotter components are not compatible with alcohol gt 10 e g methanol ethanol or organic solvents e g acetone chloroform toluene benzene Use of organic solvents voids all warranties The instrument is designed for operation in closed laboratory rooms at ambient temperature from 5 C to 40 C and maximum relative humidity 80 for temperatures up to 31 C decreasing linearly to 50 relative humidity at 40 C 4
13. h nitrocellulose with smaller membrane pore size or PVDF membrane Power Supply turns off Power Supply does not work at Use proper Power Supply low voltages inner resistance which can operate at low too high voltages e g Standard Power Pack P25 Strong increase of Blot sandwich dries out Reduce amperage soak voltage during transfer blotting paper cuts with more transfer buffer elctrodes by electrolysis Reduce transfer time membrane prewetting membrane transfer time pe buffers before use Reduce transfer time Conductivity of buffer too high Check transfer buffers Use buufers described in this manual 22 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 9 Product Information 014 100 Fastblot B33 complete system electrode surface 16 cm x 20 cm with flow through cooling incl manual 014 200 Fastblot B34 complete system electrode surface 16 cm x 20 cm without cooling incl manual 015 100 Fastblot B43 complete system with special metal electrodes platinum coated titan anode and stainless steel cathode electrode surface 16 cm x 20 cm with flow through cooling incl manual 015 200 Fastblot B44 complete system with special metal electrodes platinum coated titan anode and stainless steel cathode electrode surface 16 cm x 20 cm without cooling incl manual 015 600 Fastblot B64 complete system electrode surface 23 5 cm x 38 5 cm passive cooling incl man
14. inlet and outlet The cooling water flow should be max 0 5 1 0 min unpressurised Use no organic solvents or alcohol Best cooling is obtained using a refrigerated circulator chiller e g Code No 043 300 or 043 390 with a temperature of 5 C Attention Reduce flow rate to max 1 l min Step 13 After finishing the blot transfer switch off the current first disconnect Power Supply and Fastblot and turn off the cooling water After this take off the lid Danger High voltage Never try to detach the cables from the lid and never open the system during blotting Step 14 Remove the blot sandwich carefully and take the membrane out of the sandwich Then proceed with staining or immunoassay A Never touch the membrane with fingers without gloves Step 15 After every blotting procedure clean the plate electrodes with distilled water A paper towel can be used for drying Never use any organic solvent for cleaning carbon electrodes or housing Fastblot Instruction Manual 2009 07 13 biometra a part of Analytik Jena 5 1 2 Using a Discontinuous Buffer System The use of discontinuous buffer systems allows a higher resolution of the proteins and the transfer kinetics can be improved The method essentially resembles the blotting with continuous buffer system The main difference is that 3 different buffers are used For detail refer to chapter 7 otep 1 After the electrophoresis incubate the gel in catho
15. lactosidase MW 116 kDa 5 Myosin MW 205 kDa 50x 10 20 30 Transfer Time min Fig 1 Electrophoretic transfer of proteins SDS PAGE Acrylamide concentration 10 Nitrocellulose blotting membrane pore size 0 45 um current 5 mA per cm gel thickness of gel 1 0 mm transfer buffer Tris Glycine SDS Using B43 B44 may reduce the transfer time in the range of 10 2096 6 4 Cooling When blotting high molecular weight proteins gt 100 kDa blotting will take up to 30 min In this case or when blotting native proteins enzymes we suggest using the cooling system of the Fastblot B33 or B43 18 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 7 Buffers and Staining Destaining Solutions 7 1 Continuous Buffer Systems Transfer buffer pH 8 3 25 mM Tris base 150 mM Glycine 10 Methanol Optional Dilute the electrophoresis running buffer with one or two volumes of distilled water and add 10 20 Methanol Running buffer Laemmli System 25 mM Tris base 192 mM Glycine 0 1 96 SDS Sodiumdodecylsulphate For blotting dilute with one or two volumes distilled water Towbin buffer 50 mM Tris 96 mM Glycine pH 7 8 8 4 A Max Amperage for use with Fastblot B64 3 5 mA cm gel Fastblot Instruction Manual 2009 07 19 biometra a part of Analytik Jena 7 2 Discontiuous Buffer Systems Anode buffer 300 mM Tris HCl pH 10 4 20 v v Methanol Anode buffer II 25 mM Tri
16. ler pore size nitrocellulose membrane 0 2 um will eliminate this loss Large proteins gt 100 000 daltons denatured by SDS may transfer poorly with the addition of alcohol to the transfer buffer Alcohol increases binding of SDS proteins to nitrocellulose but decreases pore sizes in the gel Elimination of alcohol from SDS protein transfers also results in considerably diminished binding to nitrocellulose Because of the high field strength during the blotting process proteins may be transferred through the nitrocellulose membrane without binding The efficiency of binding can be increased by employing a smaller pore size nitrocellulose 6 3 Blotting Conditions The blotting conditions time current should be optimised for every protein Time and transfer rates of different proteins are summarised in Figure 1 Using the plot shown in Fig 1 it is possible to carry out a rough estimation of the transfer times e The transfer time is also influenced by the thickness and the acrylamide concentration of the gel e lf the transfer time is too long the protein may pass through the blotting membrane and is lost You can check this by adding another layer of membrane and check the second membrane also for protein after blotting Fastblot Instruction Manual 2009 07 17 biometra a part of Analytik Jena 100 Transfer Rate 1 Carbonic Anhydrase MW 29 kDa 2 Albumin egg MW 40 kDa 3 Bovine serum albumin BSA MW 67 kDa 4 B Ga
17. m or parts of those Please contact your responsible person for details 28 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 14 Note for Disposal of Electric Electronic Waste This symbol the crossed out wheelie bin means that this product should be brought to the return systems and or separate systems available to end users according to yours country regulations when this product has reached the end of its lifetime For details please contact your local distributor This symbol applies only to the countries within the EEA EEA European Economics Area comprising all EU members plus Norway Iceland and Liechtenstein Fastblot Instruction Manual 2009 07 29 biometra a part of Analytik Jena 15 EC Declaration of Conformity EU Konformitatserklarung Goettingen July 2009 im Sinne der EG Richtlinie f r die elektromagnetische Vertraglichkeit 2004 108 EG following the EC directive about the electromagnetic compatibility 2004 108 EC Hiermit erkl ren wir dass folgende Blotting Systeme Herewith we declare that the following Blotting Systems Typ type Fastblot B33 Fastblot B34 Fastblot B43 Fastblot B44 Fastblot B64 Best Nr Order No 014 100 014 200 015 100 015 200 015 600 den grundlegenden Anforderungen der corresponds to the basic requirements of EG Niederspannungsrichtlinie 2006 95 EG entsprechen EC low voltage directive 2006 95 EC Juergen Otte PhD Quali
18. mbrane are used in the same size as the gel For the further proceedings please refer to chapter 5 1 1 Preparing a Blotting Sandwich Fastblot Instruction Manual 2009 07 15 biometra a part of Analytik Jena 5 2 DNA Blotting Agarose Gels Using the Fastblot also nucleic acids can be transferred to membranes Nevertheless the use of a Vac Blot System Code No 053 000 and 053 300 is recommended 5 2 1 Sample Preparation Prior to the transfer the DNA should be pre treated in order to increase the efficiency of the blotting Denaturation 0 5 M NaOH 15 min 1 5 M NaCI Neutralisation 3 0 M NaCl 15 min 0 5 M Tris pH 7 4 Build up the blotting sandwich as described in chapter Fehler Verweisquelle konnte nicht gefunden werden but use 1 x TAE or 1 x TBE as transfer buffer and 10 layers of 0 34 mm thick blotting paper Whatman 3MM Chr paper Code No 3030700 3030704 3030861 3030917 and 3030931 or 4 layers of 1 2 mm thick blotting paper Whatman GBO005 Code No 10426981 10426994 on either side of the membrane gel sandwich For the transfer of DNA the membrane e g nylon has to be on the anode side of the gel Using 3 5 mA per cm of gel the transfer takes about 30 minutes Do not use nitrocellulose membranes because they are not stable in basic A solutions For the further proceedings please refer to chapter 5 1 1 Preparing a Blotting Sandwich 5 3 Stacked Gel transfer Two layer transfer can
19. onic Waste 29 15 EC Declaration of Conformity EU Konformitatserkl rung 30 WO CI Ds PERRO 31 2 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 1 Introduction 1 1 Field of Application The Biometra Fastblot is intended to be used for electroblotting in laboratoryies Electroblotting has become an important method transfering proteins from polyacrylamide gels onto nitrocellulose nylon or other membranes Conventional blotting tank blotting is performed in a big buffer chamber and the procedure is very buffer and time consuming The more recent method of semi dry blotting between carbon plate electrodes allows a more rapid and homogeneous transfer Pure unmodified carbon plates originally used for semi dry blotting show extensive corrosion and decomposition The electrodes used for the Fastblots B33 B34 and B64 are made of special modified carbon based material The electrodes used for Fastblot B43 and B44 are made of special modified metal All this materials are extremely resistant and allow transfer at higher currents Transfers are usually completed within 10 to 30 minutes and are uniform in all areas of the polyacrylamide gel With the semi dry blotting procedure even high molecular weight proteins gt 100 kDa are transferred from SDS polyacrylamide gels to a membrane at high currents The Fastblot is availabl
20. s HCl pH 10 4 20 v v Methanol Cathode buffer 25 mM Tris HCl pH 9 4 40 mM Capronic acid 20 v v Methanol 0 01 96 SDS alternative Cathode buffer 40 mM D L norleucine 25 mM Tris HCl pH 9 4 Dissolve norleucine with warming Stock solutions of norleucine cannot be prepared due to the limited solubility 20 Methanol A Max Amperage for use with Fastblot B64 2 5 mA cm gel 7 3 Notes for blotting using Methanol SDS Methanol max 20 v v enhances binding of proteins to nitrocellulose reduces gel swelling may reduce solubility of proteins in this case proteins are transferred less efficiently SDS max 1 w v ensures homogeneous negative charge of proteins may change antigenic properties of proteins may reduce binding of protein to the emebrane 20 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 7 4 Buffers for DNA transfer 50 x TAE Buffer Stock 1 Liter 242 g Tris base pH 8 0 57 1 ml Glacial acetic acid 100 uml EDTA 0 5 M 5 x TBE Buffer Stock 1 Liter o4 g Tris base pH 8 0 275 9 Boric acid 20 m EDTA 0 5 M 7 3 Staining and Destaining solutions Staining Solution 1 litre 2 0 g Coomassie Brilliant Blue R 250 0 5 g Coomassie Brilliant Blue G 250 425 ml Ethanol o0 ml Methanol 100 mi Acetic acid 425 ml bidest water Stir overnight filtrate before use store in dark bottle Destaining solutions acrylamide gels Fast destaining in 45 Ethanol
21. ssssesssseseeee eee nnne nnn nnne nnn a nnne nna nns 19 7 2 Discontiuous Buffer Systems cccccccccceececececeeeeeeeeceeeceeeeseeeeeaeeseeeeeeeeseeesseeeseeess 20 7 3 Notes for blotting using Methanol SDS 00 0 0 ccc ccccceeeceeeeceeeeaeeeeeeeeseeseeesseeesaees 20 7 4 Buffers for DNA transfer cece ceccc cece ce ceceeeeeececeeeeeeeeseeeeeeeesseeeseeeseeessuseseeeeseeesaeees 21 7 5 Staining and Destaining solutions 2 2 00 cece ceccceeeeeeeeeeeeeseeeeeeeeaeeeseeesseeeseeesaueesaees 21 8 Trouble Shooting eter ia ee Oe ast ee ee ree eee a 22 Fastblot Instruction Manual 2009 07 1 biometra a part of Analytik Jena 9 PIoductIBIOFmauols senatu drm a FUAN d AT Ex RN UE EUIS REM RU UU RUE 23 10 REIETENCES RI E Escort 24 11 Maintenance and Repair 220220002002000n0nn0nann nun nun nun nun nun nnne ann nun nnun nn nnunnnnn nnns 25 11 1 Cleaning and Maintenance 0220022002200200 nenn nnnnnnnnnnnennnnnnsnnnnnnnnnnn nennen 25 112 FCN A a ee ee 25 11 3 Replacement of Spare Parts u 200220sssesnnesnnennnennnennnennnnnnnnne nenn nenn nenn nennen 25 11 4 OMMEFACCESSONES oi nn nenne 25 1e SENIE E T E een 26 12 1 Instructions for return shipment cece cece eecce sees eeteeeeeeeceeeeeeeeeeeeeeeeesteeeteeeneeeaes 26 13 Equipment Decontamination Certificate eeseeeeeee eee ree enne nnn 27 14 Note for Disposal of Electric Electr
22. ted in anode buffer II Attention Avoid the inclusion of air bubbles white spots on the membrane and wear gloves when handling the membrane Step 4 Equilibrate the gel in transfer buffer for 5 min Using as discontinous buffer system the gel has to be equilibrated in the cathode transfer buffer Attention Gel equilibration time has to be consistent Shortened or extended times may affect transfer efficiencies Step 5 Place the SDS polyacrylamide gel on top of the membrane A Attention Avoid air bubbles Fastblot Instruction Manual 2009 07 11 biometra a part of Analvtik Jena Step 6 Add five 5 layers of 0 34 mm thick Blotting paper Whatman 3MM Chr or three 3 layers of 0 92 mm thick Blotting paper Whatman 17Chr or two 2 layers of 1 2 mm thick Blotting paper Whatman GB005 previously soaked with transfer buffer on top of the polyacrylamide gel DO NOT use acidic transfer buffers pH lt 3 or alkaline transfer buffers pH gt 9 B64 resp pH gt 10 Step 7 Remove air bubbles which might be included by carefully rolling out the blot sandwich with a tube or a pipette Step 8 Connect the lid with the body of the Fastblot Take care that lid and body are attached completely horizontally When handling thicker gels put a weight of 1 2 kg on top of the lid e g a beaker glass with cold water Step 9 If you have assembled the sandwich for blotting the set up should look as
23. ter is the inlet the connection with the bigger diameter is the outlet Best cooling is obtained using a refrigerated circulator chiller with a temperature of 5 C Attention Reduce flow rate to max 1 l min and use no organic solvents or alcohol Operating Conditions B44 B64 ne OT OT Max Voltage o mA 3 5 mA Max Current cm gel 8 W 10 W 50 C Max Temperature H3 10 H3 9 pH Range Electrodes B j Max Flow Rate of 0 5 1 n A 0 5 1 n A n A Cooling Water I min Unpressurised Lengthy transfer times are not recommended Do not leave the instrument unattended Joule heat can be generated rapidly during semi dry blotting Transferring longer than 2 hours can damage the unit Fastblot Instruction Manual 2009 07 7 biometra a part of Analytik Jena Do not adjust the pH of transfer buffers unless specifically indicated Follow the instructions carefully Adjustment of pH of transfer buffers when not indicated will result in increased buffer conductivity This is manifested by a higher than expected initial current output as shown by the power supply s current meter Do not use alcohol e g methanol ethanol or organic solvents for cooling or cleaning the apparatus This products are designed and certified to meet EN 61010 1 safety standards Certified products are safe to use when operated in accordance with the instruction manual This instruments should not be modified
24. ty Manager 30 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 16 Warranty This laboratory instrument is produced with the highest practical standards of materials workmanship and design The design and manufacture of parts have been conceived with one purpose to produce units which will give satisfactory service Biometra GmbH guarantees this unit to be free from defects in materials or workmanship under normal use or service for 24 month from date of shipment If during this time this unit proves defective in materials or workmanship Biometra GmbH will repair or replace it free of charge if returned to us prepaid This guarantee does not cover damage in transit damage caused by carelessness misuse or neglect or unsatisfactory performance as a result of conditions beyond our control or consequential losses as a result of failure of our product Biometra GmbH Service Department Rudolf Wissell Str 30 Rudolf Wissell Str 14 16 D 37079 Goettingen D 37079 Goettingen Tel 49 0 5 51 50 68 6 0 Tel 49 0 5 51 50 68 6 10 or 12 Fax 49 0 5 51 50 68 6 66 Fax 49 0 5 51 50 68 8 11 e mail Info biometra com e mail Service biometra com internet http www biometra de Fastblot Instruction Manual 2009 07 31
25. ty interlock Platinum coated titanium anode oafety distance pieces Plasticised carbonl cathode Plasticised carbon anode 10 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 5 Operating 5 1 Protein Blotting To determine the optimum conditions for a particular sample a time course of transfer should be performed Since many factors affect transfer e g molecular weight pl and porosity of the gel transferring for the full suggested time may not be necessary 5 1 1 Preparing a Blotting Sandwich Step 1 After finishing the electrophoresis immediately remove the gel from the glass plates and cut out from the gel the part you want to blot A Attention Wear gloves Step 2 Cut the blotting paper to the gel size and soak five 5 layers of 0 34 mm thick Blotting paper Whatman 3MM Chr or three 3 layers of 0 92 mm thick Blotting paper Whatman 17Chr or two 2 layers of 1 2 mm thick Blotting paper Whatman GBOO5 with transfer buffer Place the soaked filter paper on the anode The anode is the plate electrode on the body of the Fastblot Step 3 Cut the membrane usually nitrocellulose carefully to gel size prewet it with buffer for 5 min and place it on the filter paper prepared in step 2 A PVDF membrane has to be successively incubated in methanol 1 2 seconds in H2O 5 minutes and in transfer buffer prior to use Using a discontinuous buffer system the gel has to be prewe
26. ual Blotting Paper 10426981 Whatman GBO005 200 mm x 200 mm 1 2 mm thick 25 sheets 10 426 994 Whatman GBO005 580 mm x 580 mm 1 2 mm thick 25 sheets 3017915 Whatman 17Chr 460 mm x 570 mm 0 92 mm thick 100 sheets 3030 931 Whatman 3MM 580 mm x 680 mm cm 0 34 mm thick 100 sheets Nitrocellulose Blotting Membranes 10 401 396 Whatman Protran BA83 Pore size 0 20 um 30 cm x 3 m roll 10 401 196 Whatman Protran BA85 Pore size 0 45 um 30 cm x 3 m roll 10 401 180 Whatman Protran BA85 Pore size 0 45 um 30 cm x 60 cm 5 sheets pack 10 402 580 Whatman Protran BA85 Pore size 0 45 um 33 cm x 56 cm 5 sheets pack 10 439 196 Whatman Protran BA S 85 Pore size 0 45 um 30 cm x 3 m roll Fastblot Instruction Manual 2009 07 23 biometra a part of Analytik Jena 10 References 1 Towbin H Staehlin T and Gordon J 1979 Proc Nat Acad Sci 76 4350 4356 2 Bittner M Kupferer P and Morris C F 1980 Anal Biochem 102 459 571 3 Burnette W N 1981 Anal Biochem 112 195 203 4 Kyse Andersen J 1984 J Biochem Biophys Meth 10 203 209 24 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena 11 Maintenance and Repair 11 1 Cleaning and Maintenance Do not immerse the unit in liquid Use special care when cleaning the anode plate of B43 B44 to avoid scratching or marring the platinum Do not use abrasives or strong detergents The cathode plate stainless steel of B4
27. ument if cracks are present in the body or the safety cover or if the electrical connection cables are worn or frayed Make sure that the on off switch of the used external Power Supply is always free accessible Danger High voltage The 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 gt gt RP PP 6 Fastblot Instruction Manual 2009 07 biometra a part of Analytik Jena Power to the instrument is supplied by an external DC voltage Power Supply The output of this Power Supply must be isolated from external ground to issue that the DC voltage output floats with respect to ground All Biometra Power Supplies meet this safety requirement Never place the instrument on top of a Power Supply Do not reverse polarity of the electrodes The anode is on the body of the Fastblot and the cathode is fixed in the lid Change of polarity will result in corrosion and rusting of the stainless steel cathode or damage e g bubbling ofthe plasticised carbon electrodes Using the cooling option do not mix up the in and out plugs for the cooling water The connection for the water cooling with the smaller diame
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