User`s manual
Contents
1. cence Buffer 1x cence Buffer 10x purified Component 10 water 270 ml 27 ml 243 ml NuPage SDS MOPS Running Buffer 1x Vol of NuPage Vol of NuPage Vol of SDS MOPS SDS MOPS Run purified Running Buffer ning Buffer 10x water 1x 1000 ml 50 ml 950 ml Transfer Buffer Vol of Vol of Vol of Vol of Transfer Transfer purified methanol Buffer 1x Buffer 10x water 98 2000 ml 200ml 1600 ml 200ml TBST Vol of TBST Vol of TBS Vol of 50 1x 20x purified Tween 20 water 1000m 50 ml 950ml 1ml Ponceau S Vol of Ponceau S Vol of Ponceau S Vol TBST 1x 1x 20x 1000 ml 50 ml 950 ml Appendix Il Volumes needed for different numbers of samples No of Tray size Membrane TBST 1 Anti gels cm size body 6 Large 13 x 17 cm 50 ml 10 ul 22 4 x 15 6 22 4 x 15 6 15 x 11 4 15 x 11 4 5 5 x 9 5 Tray size Lumines cm Antibody cence Buffer 12 5 25 o m 5ml CDP Star Large 50 ml 10 ul 100 50 ul ze e o gt 15 6 Large 50 ml 10 pl 4 ml 40 ul cad a 15 6 Medium 25 ml 5 ul ae ee 30 ul 15 x 11 4 Medium 5 ul ea 30 ul 15 x 11 4 Appendix Ill Scheme for placement of gels on blot membrane Recommended scheme for placement of gels on blotting membrane 96 well plate filled with 48 duplicate samples transferred to six 17 slot gels Numbers indicate samples 1 48 M molecular size marker with undigested PrP Membrane M 1 12. and forceps Balance Dispensor for homogenization working solution 1 2 ml 96 deep well plate used as sample Master Plate PrioGENIZER homogenization device with six racks and one tray Prionics AG Product No 10000 and PrioCLIP homogenization con tainers Prionics AG Product No 10010 or FASTH MediFASTH or FASTH 2 homogeniza tion device Consul AR S A Product No 80040 82040 80020 and Prypcon homogenization containers Consul AR S A Product No 80300 or Omnisystem homogenization device Omni In ternational Inc Product No TH220P and Omni tips Omni International Inc Product No 32750 Protease Digestion m 96 well microplates 0 2 ml wells used as Digestion Plate 7 Sealing film Microplate incubator reaching at least 100 C Gel Electrophoresis 12 NuPAGE Gels 17 slots Invitrogen Product No NP0349BOX NuPAGE MOPS SDS Running Buffer Invitro gen 500 ml Product No NP0001 5 Product No NP0001 02 NuPAGE Antioxidant Invitrogen Product No NP0005 Blotting 7 PVDF membrane Immobilon P 0 45 um Millipore Product No IPVH 00010 Methanol approx 98 Transfer Buffer 10x 30 28 g Tris base 144 13 g Glycine add purified water to 1000 ml Immunological Detection Tris Buffered Saline TBS pH 7 4 8 g NaCl 0 2 g KCl 3 g Tris base Add purified water to 1000 ml adjust pH to 7 4 with HCI Tris Buffered Saline with Tween TBST TB
3. area to be used by the BSE Reference Center Note after sample collection a complete hemi section of the brain stem with an intact obex region must remain available for confirmatory testing HOMOGENIZATION Preparatory Steps Dilute 5x Homogenization Buffer Component 1 with purified water to prepare homogenization working solution Appendix l Homogenization Transfer sample to a homogenization container and determine weight on balance 0 45 0 70 g Add ten volumes of homogenization working solution w v e g 5 ml to 0 50 g brain tissue and homogenize sample using the PrioGENIZ ER Program PO PRIONICS TSE the FASTH MediFASTH FASTH 2 45 sec 5 sec 20 000 1000 rpm or the Omnisystem 60 10 sec at maximum speed homogenization device Store two 1 ml samples per homogenate in a 96 well sample Master Plate From now on each step will be done with two samples per original homogenate z PrioCLIP and Prypcon homogenization containers of samples tested TSE negative may be washed for reuse see Prio CLIP Prypcon Wash Protocol Appendix IV PROTEASE DIGESTION Following amounts are for 48 samples See Appendix II for volumes needed for samples numbers other than 48 Preparatory Steps Set the temperature of the microplate incubator to 48 1 C approx 1 hour prior to use Add 10 ul of Digestion buffer Component 2 to each well of the Digestion Plate Protease Digestion Trans
4. for guidance Transmissible spongiform encephalopathy agents safe working and the preven tion of infection Department of Health London UK can be ordered at the Stationery Office ISBN 0113221665 phone number 44 20 7873 9090 An update is available under www advisorybodies doh gov uk acdp tseguidance index htm R amp S Statements Component 1 Homogenization Buffer 5x Hazard Code This product is not classified according to EU regulations Component 2 x Xn Harmful Digestion Buffer 1x Hazard Code R22 Harmful if swallowed R36 38 Irritating to eyes and skin 23 Do not breathe gas fumes vapour spray S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S35 This material and its container must be disposed of in a safe way 36 37 Wear suitable protective clothing and gloves Component 3 Proteinase K Hazard Code This product is not classified according to EU regulations Component 4 Digestion Stop 1x Hazard Code This product is not classified according to EU regulations Component 5 Control Sample Hazard Code This product is not classified according to EU regulations Component 6 z PAGE Sample Buffer C Corrosive Hazard Code R21 Harmful in contact with skin R34 Causes burns R52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment S26 In case of contact with eyes rinse immediately with plen
5. 2 2 3 3 4 4 5 5 6 6 7 7 8 8 M 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 Gel 1 Gel 2 M 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 M 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 M 33 33 34 34 35 35 36 36 37 37 38 38 39 39 40 40 M 41 41 42 42 43 43 44 44 45 45 46 46 47 47 48 48 Appendix IV PrioCLIP Prypcon Wash Protocol General instructions Sample traceability PrioCLIP Prypcon homogenization containers must be labeled with sample number using e g a water proof pen or labels to guarantee the sample trace ability Labeling of the containers can only be removed after release of results PrioCLIP Prypcon usage traceability Homogenization containers should not be used more than 5 times PrioCLIP Prypcon have to be labeled with dashes or dots using a waterproof pen after each use Do not use hypochlorite containing disinfectants for washing Preparatory Steps Fill two vessels with sufficient amounts of de ionized water at least 25 I in order to allow com plete submersion of the PrioCLIP Prypcon during the washing steps Draining Empty containers with homogenates tested TSE negative into an autoclavable heat resistant bottle or a disposable canister flask Containers whose contents have been identi fied initial reactive must not be re used and have to be disposed of according to the na tional safety guidelines Washing Immerse the emp
6. Prionics Check WESTERN Test for in vitro detection of TSE related PrP Within the European Union this test is approved as rapid test for the BSE testing program on cattle which is set up in accordance with Regulation EC No 999 2001 Kit for 100 samples duplicate analyses Prionics AG Version 10 0 e Package Insert For in vitro veterinary diagnostic use only Store at 543 C Product No 12000 The producer of the rapid tests must have a quality assurance system in place agreed by the Community Reference Laboratory which ensures that the test performance does not change The producer must provide the test protocol to the Community Reference Laboratory Sampling tools and modifications to the rapid test or to the test protocol including sampling may only be made following advance notification to the Community Reference Laboratory CRL and provided that the Community Reference Laboratory finds that the modification does not reduce the sensitivity specificity or reliability of the rapid test That finding shall be communicated to the Commission and to the national reference laboratories Introduction Various tissues of a prion infected animal contain a pathologically altered disease specific form of the prion protein PrP The altered prion protein is de nominated PrP The normal isoform of PrP is termed PrP the cellular form of PrP PrP differs from PrP in its protease resistance Upon treatment with Proteina
7. S with 0 05 v v Tween 20 Ponceau S 20x 0 5 w v Ponceau S 5 v v acetic acid Dilute with TBST to 1x for use CDP Star concentrate Alkaline Phosphatase Substrate Applied Biosystems 12 5 mM Cat No MSC050 or Roche Diagnostics GmbH 25 mM Cat No 1759051 or CDP Star ready to use Roche Diagnostics Cat No 2041677 7 X Ray films Test Procedure Precautions National guidelines for working with prions must be strictly followed see also section Safety Regulations and R amp S Statements Appendix V The Prionics Check WESTERN must be performed in laboratories suited for this purpose Persons performing the test have to be trained gener ally i in working with prions and specifically in perform ing the Prionics Check WESTERN Samples should be considered as potentially infectious and all items which were in contact with the samples as potentially contaminated Chemical hazard data are available in section Safety Regulations and R amp S Statements Appendix V Notes To achieve optimal results with the Prionics Check WESTERN the following aspects must be considered The Test Procedure protocol must be strictly followed Pipette tips have to be changed for every pipet ting step The use of either pipette filter tips or separate pipettes for the different pipetting steps is strongly recommended In addition the accuracy of pipettes should be calibrated regularly Na tional g
8. ed pathological prion protein and the undigested normal protein If this test is to be used for screening a repeat reactive sample must be confirmed in a National Reference Laboratory using an additional confirmatory method If used for confirmation this test can only be used in conjunction with OIE CRL recommendations General Remarks Notice This manual is believed to be complete and accurate at the time of publication In no event shall Prionics AG be liable for incidental or consequential damage in connection with or arising from the use of this manual Liability Prionics AG warrants its products will meet their applicable published specification when used in accordance with their applicable instructions and within the declared products life time Prionics AG makes no other warranty expressed or implied There is no warranty of merchantability or fitness for a par ticular purpose The warranty provided herein and the data specifications and descriptions of Prionics AG products appearing in Prionics AG published cata logues and product literature may not be altered except by express written agreement signed by an officer of Prionics AG Representation oral or written which are inconsistent with this warranty or such publications are not authorized and if given should not be relied upon In the event of a breach of the foregoing warranty Prionics AG s sole obligation shall be to repair or replace at its option the appl
9. fer 100 ul mix first by pipetting up and down at least three times of each homogenate from the Master Plate to the corresponding well of the Digestion Plate with a multichannel pi pette Afterwards the Master Plate may be cov ered and stored at 20 C to 80 C for up to 12 months Add 10 ul of Proteinase K Component 3 to each well of the Digestion Plate and mix by pi petting up and down at least three times Cover the Digestion Plate with a Sealing Film Digest for 40 1 min at 48 1 C Stop the reaction by adding 10 ul Digestion Stop Component 4 Mix by pipetting up and down at least three times GEL ELECTROPHORESIS cepa Steps Mount 17 slot 12 NuPAGE gels Carefully remove the comb and white plastic foil at the bottom of the gel Heat Control Sample Component 5 to 65 3 C for 2 5 min Set the temperature of the microplate incubator to 98 4 C approx 1 hour prior to use Gel Electrophoresis Add 100 ul of PAGE Sample Buffer Component 6 to the digested homogenate in the Digestion Plate and mix by pipetting up and down at least three times Boil samples at 98 4 C for 5 min 30 s The Digestion Plate may be covered with a Sealing Film and stored at 20 C to 80 C for up to 5 days Previously prepared samples are heated to 65 3 C for 2 5 min before loading Sample loading Load 10 ul of the Control Sample in the first lane Load 10 ul of the heated samples per lane Fill
10. icable product or part thereof provided the customer notifies Prionics AG promptly of any such breach If after exercising rea sonable efforts Prionics AG is unable to repair or replace the product or part then Prionics AG shall gt O i refund to the customer all monies paid for such appli cable product or part Prionics AG shall not be liable for consequential incidental special or any other indirect damages resulting from economic loss or property damage sustained by any customer from the use of its prod ucts Prionics AG is an ISO 9001 2000 certified company Appendix Tables for preparation of working solutions Homogenization working solution Mix indicated volumes of purified pure water and 5x Homogenization Buffer Component 1 to obtain the desired volume of homogenization working solution Shelf life of homogenization working solution 1 week at 5 3 C Vol of homog Volume of Volume of enization work Homogenization purified water ing solution Buffer 5x Component 1 250 ml 50 ml 200 ml 500 ml 100 ml 400 ml 1000 ml 200 ml 800 ml PVDF Blocking Buffer Vol of PVDF Vol of Lumines Vol of Blocking Buffer cence Buffer 5x purified 1x Component 7 water 500 ml 100 ml 400 ml Luminescence Buffer Mix indicated volumes of purified water and 10x Luminescence Buffer Component 6 to obtain the desired volume of Luminescence Buffer 1x Vol of Lumines Vol of Lumines Vol of
11. ion Buffer Cap color code yellow Component 3 Proteinase K Ready to use One vial containing 4 ml of Proteinase K Cap color code white Component 4 Digestion Stop 1x Ready to use One vial containing 4 ml of Proteinase K blocker to stop proteolytic activity of the Proteinase K Cap color code red Component 5 Control Sample Ready to use One vial containing 200 ul functional control normal PrP and molecular weight markers 97 66 45 30 20 14 kD in PAGE Sample Buffer Mix before use e g by flicking the tube Component 6 PAGE Sample Buffer 1x Ready to use One vial containing 25 ml of Sample Buffer for SDS Polyacrylamide Gel Electrophoresis PAGE Contains 2 mercaptoethanol Opened vials release a bad smell However even if 100 vials are opened simultaneously in a normal aerated room air concentrations do not reach the Workplace Environmental Exposure Level of 0 65 mg m defined by the American Industrial Hygiene Association Component 7 PVDF Blocking Buffer Concentrate 5x 5x concentrate dilute before use One bottle contain ing 100 ml of concentrated Blocking Buffer to block unspecific binding sites Dilute 100 ml of Blocking Buffer with purified water to a final volume of 0 5 liter Component 8 1 Antibody 6H4 One vial containing 30 ul of monoclonal antibody to PrP mouse anti PrP IlgG1 Working dilution 1 5000 In case fluid sticks to wall or lid the tube can be centrifuged Co
12. ly cover the membrane with Saran foil Expose the membrane to an X Ray film until a strong signal of the positive control and either the background or the Proteinase K bands are visible approx 5 up to 20 min Expose longer or shorter times for optimal signal visualization Alternatively use a CCD Camera Detection Sys tem e g FluorChem Alpha Innotech Corp INTERPRETATION OF RESULTS The following figure shows the expected band patterns of BSE negative BSE positive and control samples respectively The control sample K contains the normal isoform of the prion protein PrP which is visualized via immunological detection The corre sponding diffuse band is spread from 25 35 kD due to glycosylation of PrP which causes a heterogeneous distribution K N N BSE BSE Fount strong ry ly i D 31 kDa gt i Negative samples N do not show a specific signal The 31 kD band not always visible results from unspecific binding of the secondary antibody to Pro teinase K and can be used as an orientation aid Positive samples BSEstrong BSEweak exhibit a signal consisting of three bands the top one A correspond ing to a protein with an approximate molecular weight of 30 kD The signal intensity of all bands in particular that of the lower bands B and C can be weaker than depicted here but the top band A should be clearly visible The arrow D illustrates the difference in molecular weight between digest
13. mponent 9 2 Antibody AP One vial containing 30 ul of goat anti mouse IgG AP an antibody to mouse IgG that is conjugated to alka line phosphatase Working dilution 1 5000 In case fluid sticks to wall or lid the tube can be centrifuged Component 10 Luminescence Buffer Concentrate 10x 10x concentrate dilute before use One bottle con taining 27 ml of Luminescence Buffer concentrate Dilute with purified water to 270 ml before use Additional Kit Contents Package Insert Labels for working solutions Additional Material Required The highlighted 1 items have been validated for the use with the Prionics Check WESTERN The use of different devices is in the responsibility of the user Please also see our list Prionics Check WESTERN additional material and devices for more information contact your local distributor or info prionics com General Laboratory equipment according to national safety regulations Purified water at least equivalent to Grade 3 water as defined by ISO 3696 1987 E Single channel pipette 1 10 ul Single channel pipette 10 100 ul Single channel pipette 100 1000 ul Single channel pipette 1 5 ml Multichannel pipette 0 5 10 ul Multichannel pipette 10 100 ul Pipette tips as recommended by pipette manu facturer Solution reservoirs Incubation trays 15 ml conical tubes 50 ml conical tubes Homogenization Cutting tool
14. se K PrP is degraded while PrP is reduced from its original size of 32 35 kD to a smaller size of 27 30 kD The remaining protease resistant PrP fragment is referred to as PrP27 30 The Prionics Check WESTERN achieves its high precision and reliability by monitoring three independ ent criteria protease resistance glycosylation pattern and lower molecular weight of the protease resistant PrP fragment 27 30 kD compared to normal undigested PrP The uniqu e properties of the buffer solutions used in Prionics Check WESTERN and the high affinity of the antibody allow that the test can be performed directly with tissue homogenates combining the reliability of the Western blotting procedure with the speed needed for mass screening The Prionics Check WESTERN was the first BSE test kit to be approved by the Swiss authorities in 1998 In 1999 it was officially acknowledged by the EU as the only test to achieve 100 sensitivity and 100 specificity without retesting Test Principle 1 SAMPLING HOMOGENIZATION Brainstem fay Homogenization a container Obex CONTROL HEALTHY mo digestion Proteinase K 4 ly a After Sample Collection and Registration samples are analyzed with the Prionics Check WESTERN The Prionics Check WESTERN follows a five step proto col consisting of Homogenization Protease Digestion Gel Electrophoresis Blotting and Immunological Detection One person can proce
15. ss 100 samples duplicate assays within 6 8 hours Samples are collected registered and a homogenate is prepared from a defined piece of brain tissue Treatment with Proteinase K degrades PrP com pletely while PrP is reduced to the 27 30 kD m ment The proteolytic reaction is stopped and PrP gt detected in the Prionics Check WESTERN assay Digested homogenates are subjected to gel electro phoresis and Western blotting The blot membranes are incubated with a monoclonal antibody with high affinity for PrP for the detection of protease resistant PrP The signal is visualized using the secondary antibody alkaline phosphatase AP conjugate Kit Components Shelf life of all un opened components is 1 year after production if stored at 5 3 C See kit label for actual expiry date The shelf life of diluted opened or recon stituted components is noted below when appropriate Chemical hazard data are available in section Safety Regulations and R amp S Statements Appendix V Component 1 Homogenisation Buffer Concentrate 5x 5x concentrate dilute before use One bottle contain ing 200 ml of 5x concentrated Homogenization Buffer Prepare 1x homogenization working solution by mixing 1 part Homogenisation Buffer 5x with 4 parts purified water Shelf life of the homogenization working solution 1 week at 543 C Component 2 Digestion Buffer 1x Ready to use One vial containing 4 ml of Digest
16. stain bound proteins with 1x Ponceau S Label the position of the size markers Destaining is performed with TBST until the red color has disappeared approx 2 x 1 min IMMUNOLOGICAL DETECTION Blocking Incubate the membrane in a plastic incubation tray with 50 2 ml of 1x PVDF Blocking Buffer Component 7 see Appendix I for the dilution table for 35 5 min at 22 3 C on a rocking plat form with gentle agitation 1 Antibody Dilute 10 ul of 1 Antibody 6H4 Component 8 in 50 2 ml of TBST 1 5000 dilution add to mem brane and incubate for 60 5 min at 22 3 C or alternatively for 12 18 h at 5 3 C with gentle agitation on a rocking platform Wash membranes 3x for approx 5 min with TBST 2 Antibody Dilute 10 ul of 2 Antibody AP Component 9 in 50 2 ml of TBST 1 5000 dilution Incubate for 30 1 min at 22 3 C with gentle agitation Wash membranes 5x for approx 5 min with TBST Detection Equilibrate membrane for 5 10 min in 50 2 ml 1x Luminescence Buffer Component 10 see Appendix for the dilution table Dilute 100 ul CDP Star 12 5 mM 50x or 50 ul 25 mM 100x in 5 ml 1x Luminescence Buffer Place the membrane on a glass plate Distribute 5 ml of the diluted CDP Star solution or 5 ml of the ready to use solution evenly over the mem brane and incubate for 5 1 min at 22 3 C Remove excess liquid Remove remaining liquid from the membrane with a soft Kleenex tissue and immediate
17. ty PrioCLIP Prypcon in a vessel with de ionized water rinse thoroughly Inspect the homogenization containers visually for possible damage and tissue contamination during transfer from vessel one to vessel two Discard any damaged or contaminated PrioCLIP Prypcon homogenization containers Submerge containers and incubate at least 30 min at 2243 C Drying Take the PrioCLIP Prypcon out of the vessel shake out remaining water and let them dry com pletely at 22 3 C Alternatively PrioCLIP Prypcon can be dried in a heating drying oven Place the containers on a heat resistant surface heat them for 2 hrs at 85 5 C and dry over night at approx 50 C ina drying oven Repeat heating step 2 hrs 85 5 C Visually check PrioCLIP Prypcon Discard containers that are damaged or contain remaining fluid or tissue Now PrioCLIP Prypcon are ready for re use Waste disposal Homogenates and washing solutions have to be disposed of according to national safety guidelines A detailed PrioCLIP Prypcon wash protocol includ ing pictures can be requested at info prionics com Appendix V Safety Regulations and R amp S Statements Safety Regulations 1 National Safety Regulations must be strictly followed 2 ACDP guidelines Laboratories MUST adhere to National Safety Regula tions but the following information published by the Advisory Committee on Dangerous Pathogens ACDP is available
18. ty of water and seek medical advice S35 This material and its container must be disposed of in a safe way S36 37 39 Wear suitable protective clothing gloves and eye face protection S45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible Component 7 PVDF Blocking Buffer Concentrate 5x Hazard Code This product is not classified according to EU regulations Component 8 1 Antibody 6H4 Hazard Code This product is not classified according to EU regulations Component 9 2 Antibody AP Hazard Code This product is not classified according to EU regulations Component 10 Luminescence Buffer Concentrate 10x Hazard Code This product is not classified according to EU regulations Prionics AG Wagistrasse 27a CH 8952 Schlieren Zurich Switzerland Tel 41 44 200 2000 Fax 41 44 200 2010 www prionics com info prionics com For our distribution network please refer to www prionics com Fitness for purpose validated and certified by OIE Registration number 20080102
19. uidelines apply Separate solution reservoirs must be used for each reagent Kit components must not be used after their expiry date or if changes in their appearance are observed Kit components of different kit lot numbers must not be used together Non disposable cutting tools and forceps must be decontaminated according to guidelines en forced by national authorities When the PrioGENIZER is used for homog enization only program PO PRIONICS TSE must be used for homogenization of brain tissue SAMPLING AND HOMOGENIZATION Take 0 45 0 70 g nervous tissue from the preferred area of the left or the right side of the brainstem with e g a scalpel Sampling and laboratory testing must follow the Regulation EC No 999 2001 Chapter C which refers in terms of collection of samples to the latest edition of the Manual Standards for Diagnostic Test and Vac cines of the International Office of Epizootic Diseases OIE stating The preferred sample for immunoassay should be at or as close to the obex as possible but no further than 1 5 cm anterior to the obex The picture below shows the sampling area within box 4 Medulla oblongata The tissue sample is an approx 8 cm long piece of brainstem cervical spinal cord For a detailed sampling protocol contact info prionics com 1 spinal cord N brain ee obex region aN area to be used for Prionics Check WESTERN testing 5
20. up inner and outer chamber with 1x NuPAGE SDS MOPS Running Buffer and add 500 ul Nu PAGE Antioxidant to the inner chamber only Electrophoresis Run loaded gels at 200 V until the dye front is about 1 2 cm from the bottom of the gel approx 30 min BLOTTING Preparatory Steps Wet the PVDF membrane Millipore Immobilon P 0 45 um 13 x 17 cm in methanol approx 98 for a few seconds Equilibrate the mem brane for at least 10 min in 1x Transfer Buffer see Appendix II for volumes needed Fill transfer unit with pre chilled 5 3 C 1x Transfer Buffer Blotting Sandwich assembly Place membrane on Whatman Paper moistened with 1x Transfer Buffer or purified water Open plastic frame of NUPAGE gel Remove the top part of the gel containing the slots and the bottom part below the dye front Place the gel on the membrane avoid air bubbles Up to 6 gels can be placed on one membrane of the above size see Appendix III Overlay gels with moistened Whatman Paper place sponge on top Close transfer cassette and place in transfer unit Proteins are negatively charged and move to wards the positive red pole of the transfer unit Make sure that the cassette is inserted with the PVDF membrane towards the positive pole and the gels towards the negative pole Sponge Filter paper PVDF Blotting direction Transfer at 150 V for 602 min at 5 3 C with continuous cooling Remove the membrane and
Download Pdf Manuals
Related Search