User Manual - BioCat GmbH
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1. Equilibration Virus Binding To be done with a ring or clamp stand Viral Purification Elution gt Buffer i j x Optional Ready To Use Viral Particles Figure 1 Experimental Flow Chart Page 3 of 19 Viral Purification Handbook Add N Pure Adenovirus Purification Kit Add N Pure Adenovirus Purification Recombinant adenovirus is the most efficient viral system developed to date with regards to gene siRNA and miRNA delivery for in vitro and in vivo ap plications However high titers of purified recombinant adenovirus are required for many applications especially in vivo gene delivery Traditionally purification of re combinant adenovirus is achieved by Cesium chloride density gradient ultra cen trifugation This process is technically demanding time consuming and requires the availability of ultra centrifugation equipment Thus there has always been a need for the development of a simpler and more efficient adenovirus purification method to support the increasing range of adenoviral applications Add N Pure adenovirus purification kit is a chromatography based system for adenovirus purification and concentration The kit represents a significant improvement to the traditional Cesium chloride ultra centrifugation method spe cifically in terms of easy manipulation reliability and efficiency In fact the entire Add N Pure purification p2. gt Table 1 Kit Components Maxi 2X Maxi 5X Giga 2X E Component Cat A902 Cat A905 Cat A910 10X Preparation Buffer 10ml 25ml 30ml Equilibration Buffer 10ml 25ml 20ml Wash Buffer 80ml 2X 100ml 80ml Elution Buffer 20ml 50ml 20ml 10ml Syringe 2X 5X 2X 20ml Syringe 2X 5X 2X Syringe Filter 4X 10X 6X Add N Pure 2X 5X 2X Purification Unit Note The Add N Pure has been tested for single use only Additional Materials Required General Lab Equipment e Ring stand with clamps e Ethanol dry ice bath e 37 C water bath e Centrifuge Fluid Collection e 150 500ml beakers for flow through waste collection e Sterile 15ml and 50ml conical tubes Adenovirus Production e Tissue culture plates and flasks e DMEM 10 fetal bovine serum Page 5 of 19 Viral Purification Handbook Protocol The Add N Pure filter is designed for single application only Each filter included in the kit has the capacity of binding and purifying recombinant adenovirus from 5X10cm to 5X15cm dishes of packaging 293 cells with complete cytopathic effect CPE Overall pus 5 T 1 lt a maximum of 50ml viral supernatant can be processed with Cat A902 and A905 kits For the Giga Add N Pure purification unit Cat A910 150ml of viral supernatant is its capacity limit A Adenovirus Production 1 To amplify adenovirus we recommend growing 293 cells in one well of a 6 wel
3. abm Viral Purification Handbook Add N Pure A902 A905 A910 PuRetro G171 G172 G173 Buffer Exchange 6130 www abmGood com WoD POOL QD MMM Table of Contents Introductory Notice to the Purchaser Technical Support Biosafety Diagrams and Charts Protocol at a Glance Add N Pure Adenovirus Purification Kit Contents Additional Materials Required Protocol PuRetro Lentivirus and Retrovirus Purification Kit Contents Additional Materials Required Protocol Viral Stock Buffer Exchange Protocol Troubleshooting References Contact Information Viral Purification Handbook nn A 10 11 11 12 15 15 17 18 19 Notice to the Purchaser The products are for research use only Use in and or for diagnostics and therapeutics is strictly prohibited By opening and using the product the purchaser agrees to the following The components in this kit may not be distributed resold or modified for resale without prior written approval from Applied Biological Materials ABM Inc If you do not agree to the above conditions please return the UNOPENED product to ABM Inc within ten 10 days of receipt for a full refund The information in this document is subjected to change without notice and should not be construed as a commitment by ABM Inc or its affiliated corporations In no event shall ABM Inc or its affiliated corporations be liable for incidental or con
4. sequential damages in connection with or arising from the use of this manual and product ABM Inc products are warranted to meet our QC testing standards at the time of shipment Notice of problematic products must be made to ABM Inc within 15 days of receipt of the product This product warranty limits ABM Inc s liability to the replacement of the product only Technical Support For more information on ABM Inc products please visit our website http www abmGood com For additional information or technical assistance please call or e mail us at Applied Biological Material Inc Phone 604 247 2416 1 866 757 2414 Fax 604 247 2414 E mail technical abmGood com Page 1 of 19 Viral Purification Handbook Biosafety Recombinant adenoviral retroviral and lentiviral vectors have been designated as Level Il biological agents by the Centers for Disease Control of USA and other regulatory bodies of different jurisdictions worldwide Therefore all pub lished BL 2 guidelines with proper waste decontamination should be strictly followed In addition exercise extra caution when creating recombinant virus carrying poten tially harmful or toxic genes e g activated oncogenes For more information about the BL 2 guidelines and handling refer to the document Biosafety in Microbiological and Biomedical Laboratories 4th Edition published by the Centers for Disease Control CDC This document may be downloaded
5. Load the viral supernatant onto the PuRetro Purification Unit Place the feed tube into the conical tube containing the unpurified viral superna tant and pull up the syringe plunger to withdraw Multiple withdrawals may be required to pass all the viral supernatant through the filter Dispense the supernatant through the PuRetro Purification Unit at a rate of 3ml min ap proximately 1 drop per second Note To avoid dispensing air through the filter unit in this and the following step leave a small volume of supernatant in the syringe This will not affect the purification process 7 Wash the PuRetro unit with 10ml Wash Buffer Place the feed tube into the tube containing Wash Buffer and pull up the syringe plunger for with drawal Dispense through the PuRetro Purification Unit at a rate of 3ml min approximately 1 drop per second B Virus Elution 8 Remove the plunger from a sterile 10ml syringe provided in the kit 9 Remove the filter from the PuRetro Purification Unit Attach the filter to the sterile 10ml syringe 10 Add 3ml of Elution Buffer to the syringe column If the Giga purification unit is used or the elution is to undergo buffer exchange for in vivo applica tions or higher titer concentration up to 10ml Elution Buffer can be used to increase the elution efficiency 11 Replace the plunger in the syringe Page 13 of 19 Viral Purification Handbook Protocol 12 13 14 Elute and dis
6. antibodies Caspase 3 Caspase 3 knockdown in 293 cells transfected 293 cell lysates at different amount of total with 3 different RNAi oligoes proteins ug probed with Anti f actin Loading control was probed with f tubulin antibody G046 antibody G098 Receive an instant 40 discount on the antibodies above using promotion code abmBO1 at ot The Source for any Antibody siRNA and Viral Vector The Depot for PCR qPCR and Transfection Reagents 8 _ nad GET owes 89 putos y DEPT My Ae vit Men PIO Lent search wade S 9 god ore d Gene Name nc 18 3 p optet ws ni j odie jar e292 cof ag EO aN A v Sm e ood gases ea ano o 4 n socio Y M 6 C2 S we SUPE pn ere pm 38 52 A H Y wore Comit goo els i i au a x V eau M M we P Y 9
7. from the Web at the following address http www cdc gov od ohs biosfty bmbl4 bmbl4toc htm It is also important to consult with the health and safety officers for guidelines at your institution regarding the use of recombinant viruses Always follow standard microbiological practices which include Wear gloves and lab coat at all times e Always work with pseudoviral particles in a Class 11 culture facility All virus purifi cation work should be done in a biosafety cabinet excluding centrifugation and water bath steps All procedures are performed carefully to minimize splashes or aerosols e Work surfaces are decontaminated at least once a day and after any spill of vi able material e All cultures stocks and other regulated wastes are decontaminated be fore disposal by an approved decontamination method such as autoclaving Follow federal state and local regulations for disposal of potentially biohazardous wastes and handle all recombinant viruses in compliance with established institu tional guidelines Since safety requirements for use and handling of recombinant viruses may vary at individual institutions we recommend consulting the health and safety guidelines and or officers at your institution Viral Purification Handbook Page 2 of 19 Protocol at a Glance Harvest Virus Clear Virus Supernatant Pre Filtration Sample Preparation
8. not performed Otherwise perform this step after buffer exchange Step 12 Page 16 Viral Purification Handbook Page 8 of 19 Add N Pure Protocol 22 The eluted adenovirus is in a salt buffer but can be used directly for in vitro target cell transduction if the viral dilution is over 5X i e 1 0ul of viral stock to Aul culture medium For higher titers of viral stock or viral dilutions less P e m d m c than 5X during transduction a buffer exchange is required make the viral stock suitable for such applications This can be easily performed using ABM Inc s viral buffer exchange kit Cat G130 on page 15 23 For higher titers long term storage of the virus and proper tonicity for in vivo applications we recommend buffer exchange of the eluted ad enovirus into 1X Storage Buffer using ABM Inc s Buffer Exchange kit Cat G130 24 1X Storage Buffer 2 5 glycerol w v 25mM NaCl and 10mM Tris HCI pH 8 0 Hoganson et al 2002 25 Store final viral stock aliquots at 70 C in a non frost free freezer Avoid repeated freeze thaw cycles F Determining Adenoviral Titer There are three protocols for determining adenoviral titer 26 Plaque Assay approx 2 3 weeks in duration 27 End Point Dilution Assay approx 10 days in duration 28 OD Assay approx 1 hour in duration Methods 1 and 2 are biological assays they measure the number of infectious viral particles Method 3 is
9. size of ultracentrifugation tubes Reports have also stated that the ultracentrifugation process has some detrimental ohysical effects on the recombinant virus Due to limitations on the existing technique there is great necessity for a quick and efficient method to purify and concentrate recombinant retrovirus and lentivirus Scientists at ABM Inc have had years of experience with ion exchange based filter membranes and have successfully developed PuRetro the most effec tive retroviral and lentiviral purification method based on this technology Viral Purification Handbook Page 10 of 19 Kit Contents This method proves to be fast efficient and reliable Moreover it is capable of conveniently processing large volumes of viral supernatant up to 150ml using the Giga kit Depending the amount of viral supernatant input and the final elu tion amount of viral stock a 50X to 150X fold increase in viral concentration can be achieved using this method Table 2 Kit Components Maxi 2X Maxi 5X Giga 2X Component Cat G171 Cat G172 Cat G173 Equilibration Buffer 10ml 25ml 20ml Wash Buffer 20ml 50ml 20ml Elution Buffer 20ml 50ml 2X10ml 10ml Syringe 2X 5X 2X 20ml Syringe 2X 5X 2X Syringe Filter 4X 10X 6X PuRetro 2X 5X 2X Purification Unit Note The PuRetro has been tested for single use only Additional Materials Required General Lab Equipment e Ring stand with clamps e Eth
10. sterile conical tube 8 Add 10X Preparation Buffer to the supernatant The 10X Preparation Buf fer should be 1 9 of the supernatant volume Mix gently and thoroughly by gt m c inverting the tube 5 6 times 9 Using a 20ml syringe complete a pre filtration step for the superna tant with syringe filter included in the kit For the Giga kit viral supernatant over 50ml should be split up between two syringe filters Pre filtration will de crease viscosity and improve the flow rate of the sample during virus purifica tion steps Note The syringe filter is a low protein binding type to avoid loss of recombi nant virus C Adenovirus Purification 10 Clamp the syringe column of the Add N Pure Purification Unit onto a ring clamp stand Place a beaker under the filter unit for waste collection Alternatively a syringe pump can be used for the following procedures 11 Equilibrate an Add N Pure Purification Unit with 5ml of Equilibration Buffer or a Giga Add N Pure Purification Unit with 10ml of Equilibration Buf fer Place the feed tube into a 50ml conical tube containing Equilibration Buffer and pull up the syringe plunger for withdrawal of the desired amount of buffer Dispense the buffer through the Add N Pure Purification Unit by pressing down lightly on the syringe plunger The buffer should flow through the filter unit at a rate of 3ml min approximately 1 drop per second Note Avoi
11. than retrovirus Furthermore lentivirus has the ability to transduce more cell types than recombinant retrovirus including many primary cell types Cells that cannot be transduced by recombinant retrovirus are often receptive to recombinant lentiviral transduction This has led to numerous and diverse applications of recombinant len tivirus for both in vitro and in vivo gene delivery Both recombinant retrovirus and lentivirus can be produced by transient transfection from which viral supernatant can be collected and used for target cell transduction The titer of viral supernatant by transient transfection is approximately 10 cfu colony forming unit ml which is sufficient for the generation of most stable cell lines in vitro However there are applications that require higher purity and titers higher than 10 cfu ml These applications include experiments demanding higher gene transduction efficiency and in vivo gene delivery In addition crude viral su pernatants are not suitable for in vivo administration due to various contaminants contained in cell culture supernatant Thus the viral supernatant needs to be further concentrated and purified before use Traditionally both recombinant retrovirus and lentivirus have been concentrated via ultracentrifugation Although the method works well it requires specific ultracentrifugation equipment and it is technically demanding In addition the total viral supernatant volume is limited to the
12. 33 8037 Demmer W and Nussbaumer D 1999 Large scale Membrane Adsorbers J Chromatogr A 852 73 81 Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D and Na Idini L 1998 A Third Generation Lentivirus Vector with a Conditional Packaging System J Virol 72 8463 8471 Graham R L Smiley J Russel W C and Narin R 1977 Characteristics of a human cell line transformed by DNA from human adenovirus type 5 J Gen Virol 36 59 72 Hoganson D K Mq J C Asato L Ong M Printz M A Huyghe B G Sosnowshi B A and D Andrea M J 2002 Development of a stable adenoviral vector formulation Bioprocessing J 1 1 43 48 Hutchins B 2002 Development of a reference material for characterizing adeno virus vectors Bioprocessing J 1 1 25 28 Lochmuller H Jani A Huard J Prescott S Simoneau M Massie B Karpati G and Acsadi G 1994 Emergence of Early Region 1 Containing Rep lication Competent Adenovirus in Stocks of Replication Defective Adenovirus Recombinants Delta El Delta During Multiple Passages in 293 Cells Hum Gene Ther 5 1485 1491 Wang l 1 and Huang l l 2000 Adenovirus Technology for Gene Manipulation and Functional Studies Drug Discovery Today 5 10 16 Wivel N A 1999 Adenoviral Vectors In The Development of Human Gene Ther apy T Friedmann ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press pp 87 110 Yama
13. a physical assay it measures the concentration of viral DNA and viral protein and therefore does not distinguish between infectious and non infectious viral particles For detailed information about adenoviral titer assay please refer to our online tech nical support htto www abmgood com TechSupport adeno vec ph G Transduction of Target Cells For detailed information about target cell transduction with recombinant adenowvirus refer to our adenoviral expression manual http www abmgood comj viralexp Adeno 17620Expresion 7620System pdf Page 9 of 19 Viral Purification Handbook PuRetro Lentivirus amp Retrovirus Purification Kit PuRetro Lentivirus and Retrovirus Purification Recombinant retrovirus is a widely used vehicle for successful stable expression of any active dividing cell type Mann et al 1983 Miller amp Buttimore 1986 Retrovirus can efficiently integrate into the host genome giving rise to per manent and stable gene expression During cell division the nuclear membrane disintegrates allowing retroviral DNA to access host genome Because recombinant retrovirus cannot actively pass through the nuclear membrane the transduction ef ficiency of target cells with recombinant retrovirus is low especially in slow dividing primary cells Recently recombinant lentivirus a new type of retrovirus has been devel oped to transduce both dividing and non dividing cells at a much greater efficien cy
14. anol dry ice bath e 37 C water bath e Centrifuge Fluid Collection 150 500ml beakers for flow through waste collection e Sterile 15ml and 50ml conical tubes Adenovirus Production e Tissue culture plates and flasks e DMEM 10 fetal bovine serum Page 11 of 19 Viral Purification Handbook Protocol The following purification protocol is applicable for both recombinant retrovirus and lentivirus that have been pseudotyped with G glycoprotein gene from Vesicular Sto matitis Virus VSV G during viral production For the production of lentivirus and ret rovirus please refer to a manual from any commercial source relating to lentiviral or retroviral expression systems or refer to our manuals in the following web links Lentivirus Production http www abmgood com viralexp Lenti Easy His7620Expressionze20System p df Retrovirus Production http www abmgood com viralexp Retro Easy 20Expression 20System pdf A Lentivirus or Retrovirus Purification The PuRetro filter is designed for single application only A maximum of 50ml viral supernatant can be processed with Cat 171 and G172 Maxi kits while 150ml of viral supernatant is the capacity limit for the Giga PuRetro Purification Unit Cat G173 1 Clear the viral supernatant by centrifugation at 2 000g for 10 min utes 2 Transfer all viral supernatant to a new 50ml conical tube 3 Using a 20ml syringe complete a pre filtration step for the supe
15. card the first 1 0ml of Elution Buffer into a waste container The first 1 0ml is largely comprised of Wash Buffer and should not be kept Leave the rest of the buffer in the syringe column Incubate the filter at room temperature for 5 minutes Rest filter on top of a sterile 15ml conical tube to catch any premature elution Elute the rest of the Elution Buffer at a rate of 1 0ml min approximately 1 drop every 3 seconds Use the residual air in the syringe to help expel remaining Elution Buffer from the filter The eluted buffer contains the purified recombinant lentivirus or retrovirus C Post Purification Notes 15 16 Filter sterilize viral stock post purification using a low protein binding syringe filter unit provided in the kit The eluted recombinant retrovirus and lentivirus is in a salt buffer but can be used directly for in vitro target cell transduction if the viral dilution is over 5X i e 1 0ul of viral stock to Aul culture medium For higher titers of viral stock viral dilutions less than 5X during transduction long term storage of the virus or proper tonicity for in vivo applications a buffer exchange is required This can be easily performed using ABM Inc s Viral Buffer Exchange Kit Cat G130 on page 15 17 1X Storage Buffer 1X PBS buffer pH at 7 2 18 Store final viral stock aliquots at 70 C in a non frost free freezer Avoid repeated freeze thaw cycles D Determining Recombinant Lentivirus an
16. cup on 9 Check to make sure the hooks are in the notches on the sample reservoir Return the exchange device to the centrifuge and spin at 3 000g for 3 minutes to collect viral stock Note Ensure the centrifuge is counterbalanced 10 Remove the device and unscrew the concentrate cup 11 Attach the 0 2um syringe filter onto the 3ml syringe provided in Cat G130 kit 12 Transfer viral stock from the concentration cup to the 3ml syringe and push through the 0 2um syringe filter for sterilization 13 The viral stock is now ready for target cell transduction or aliquot for long term storage at 80 C Alternatively a small aliquot can be used for titer assay using established protocols or information provided on page 9 for O C Men QO LL a adenovirus and page 14 for retrovirus and lentivirus es Rees Clee Orientation in Centrifuge Membrane Paddle m Sample Reservoir Filtrate Receiver Correct Paddles oriented towards centre of rotor Cup and Cap E 2 Concentration Filtrate Reciever Wu E Incorrect Paddles oriented improperly Figure 2 Buffer Exchange filter components and operation Viral Purification Handbook Page 16 of 19 Troubleshooting Problem Pre filtration filter clogged Possible Cause High amount of cellular debris Air was pushed into the filt
17. d Retrovirus Titer For detailed information about recombinant lentivirus and retrovirus titer assay please refer to our lentivirus and retrovirus expression manuals online Lentivirus http www abmaood comy viralexp Lenti Easy His 620Expression e620System p df Retrovirus http www abmgood com viralexp Retro Easy 20Expression 20System pdf E Transduction of Target Cells For detailed information about target cell transduction using recombinant len tivirus and retrovirus please refer fo our manuals with the links above Viral Purification Handbook Page 14 of 19 Viral Stock Buffer Exchange Viral Stock Buffer Exchange Cat G130 Cat G130 is supplied separately from the recombinant viral purification kits All recombinant viral purification kits do not contain the viral stock buffer exchange kit The eluted recombinant virus is in a salt buffer but can be used directly for in vitro target cell transduction if the viral dilution is over 5X i e 1 0ul of viral stock to 4ul culture medium For higher titers of viral stock or viral dilutions less than 5X during transduction a buffer exchange is required to make the viral stock suitable for such applications This can be easily performed using ABM Inc s viral buffer exchange kit Cat C130 The exchange filter included in the kit is applicable for all recombinant virus but the exchange buffer differs a Adenoviral storage bu
18. d dispensing air through the Add N Pure Purification Unit The presence of air in the filter may cause uneven distribution of solutions in the membrane and alter the efficiency of adenovirus purification Remove the filter first before dispensing the residual air from the syringe 12 Load adenoviral supernatant into the Add N Pure Purification Unit Place the feed tube into the conical tube containing the unpurified viral su pernatant and pull up the syringe plunger to withdraw Multiple withdrawals may be required to pass all the viral supernatant through the filter Dispense the supernatant through the Add N Pure Purification Unit at a rate of 3ml min approximately 1 drop per second Note To avoid dispensing air through the filter unit in this and the following step leave a small volume of supernatant in the syringe This will not affect the purification process Page 7 of 19 Viral Purification Handbook Protocol 13 Wash the Add N Pure Purification Unit with Wash Buffer The volume of the Wash Buffer should equal the volume of the original culture medium However Wash Buffer volumes over 40ml are unnecessary Wash Buffer vol umes of 40ml is enough when input viral volume is over 40ml Place the feed tube into the 50ml conical tube containing adequate volume of Wash Buffer and pull up the syringe plunger for withdrawal Multiple withdrawals may be required to pass all the Wash Buffer through the filter Dispense th
19. dda K McCarty D M Madden V J and Walsh C E 2003 Lentivirus Vec tor Purification Using Anion Exchange HPLC Leads to Improved Gene Transfer Bio techniques 34 1074 1080 Viral Purification Handbook Page 18 of 19 Contact Information Applied Biological Materials Inc Website www dbomGood com Phone 8 30am 4 30pm PST M F Toll Free 1 866 757 2414 Local 604 247 2416 Fax 604 247 2414 24Hr Address Suite 8 13520 Crestwood Place Richmond BC Canada V6V 2G2 Distributors North America Canada Applied Biological Materials Inc Tel 604 247 2416 1 866 757 2414 Fax 604 247 2414 www abmqGood com Email General Information info abmGood com Order Products order abmGood com Technical Support technical abmGood com SIRNA sIRNA abmGood com Business Develooment bd abmGood com United States Applied Biological Materials Inc Tel 604 247 2416 1 866 757 2414 Fax 604 247 2414 www abmqGood com International Belgium France Germany Gentaur Gentaur BioCat GmbH Tel 32 2 732 5688 Tel 01 43 25 01 50 Tel 49 0 6221 714 1516 Fax 32 2 732 4414 Fax 01 43 25 01 60 Fax 49 0 6221 714 1529 www gentaur com www gentaur com www biocat com India Israel Japan G Biosciences BioConsult Cosmo Bio Co Ltd Tel 0120 432 3330 Tel 972 0 2 566 7043 Tel 03 5632 9610 9620 Fax 0120 432 3299 Fax 972 0 2 566 2790 Fax 03 5632 9619 www GBiosciences com www bioconsult co
20. er after being wetted Solution Avoid disturbing the cellular debris pellet after centrifugation Avoid pushing air into a wet filter Purification filter is clogged High amount of cellular debris Complete the pre filtration step Low virus yield Air entered the filter of the purification unit Flow rates for loading and washing were too fast Elution rate was too fast Remnants of Elution Buffer were left behind in the filter Procedure was not completed properly The inserted gene is toxic to 293 cells Avoid pushing air through the filter Slow the flow rate to 3ml min 1 drop second Slow the flow rate to 1 0ml min 1 drop seconds Expel all of the buffer with the residual air in the syringe Read and follow the protocols carefully Use an inducible system or ABM s resistant packaging 293 cells High amount of cellular DNA Excess of cellular DNA Decrease the input amount of viral supernatant Page 17 of 19 Viral Purification Handbook References Buchschacher L Jr and Wong Staal F 2000 Development of Lentiviral Vectors for Gene Therapy for Human Diseases Blood 95 2499 2504 Burns J C Friedmann T Driever W Burrascano M and Yee J K 1993 Vesicular Stomatitis Virus G Pseudotyped Retroviral Vectors Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells Proc Natl Acad Sci USA 90 80
21. ffer 2 5 glycerol w v 25mM NaCl and 10mM Tris HCI pH 8 0 Hoganson et al 2002 b Lentiviral Retroviral storage buffer 1X PBS buffer pH at 7 2 Protocol 1 Insert the paddle firmly into the bottom of the sample reservoir The hooks on the top part of the paddle must rest firmly in the notches on the top of the sample reservoir For best alignment turn the reservoir upside down on the bench top and gently press the paddle into place 2 Attach the filtrate receiver to the bottom of the sample reservoir 3 Top up the eluted viral stock to 15ml using the exchange buffer pro vided 4 Transfer all the diluted viral samples into the non membrane side of the sample reservoir Attach reservoir cap 5 Place the buffer exchange unit into a centrifuge unit that accepts standard 50ml tubes Note Ensure the membrane paddle is parallel with the center of the centri fuge Figure 2 6 Spin at 3 000g for 45 minutes to achieve desired viral stock volume 2 5ml Note Ensure the centrifuge is counterbalanced 7 Add another 10ml exchange buffer to the non membrane side of the sample reservoir and spin again at 3 000g for 35 minutes or more until the volume within the sample reservoir is approximately 1 0 1 5ml Note Ensure the centrifuge is counterbalanced Page 15 of 19 Viral Purification Handbook Protocol 8 After centrifugation remove the filtrate receiver from the unit and screw the concentration
22. il www cosmobio co jp South Korea Taiwan United Kingdom CMI Biotech Interlab Co Ltd NBS Biologicals Ltd Tel 02 444 7101 Tel 886 2 2736 7 100 Tel 44 0 1480 433875 Fax 02 444 7201 Fax 866 2 2735 9807 Fax 44 0 1480 459868 www cmibio com www interlab com tw www nbsbio co uk Page 19 of 19 Viral Purification Handbook abm 1 Antibody for Less Than 150 His Antibody HA Antibody Tag Antibodies ii Most comprehensive source n r for Tag antibodies including His HA Myc GST VSVG VS D Tag and many others His Western HisImmunocytochemistry Western Immunocytochemistry Fig Lane 1 A Untransfected 293 cells Lane 2 B 293 Cells transfected with His or HA tag constructs 1 2 118kD Signal Antibodies it M Ai 47kD Over 500 signal antibodies prid BE including rare phosphorylated cruor in antibodies 26kD 40 discount apply to Cat from YO1 1001 to YO21320 T Blocking Peptide pam Blocking Peptide Western blot analysis of extracts from K562 cells using GATA 1 phospho Ser142 antibody y011041 Immunohistochemical analysis of paraffin embedded human breast carcinoma tissue using GATA 1 phospho Ser142 antibody y011041 Control Non specific RNAiII RNAiIII 50 25 12 5 6 25 25 Tubulin Loading Control Antibodies All loading control antibodies including GAPDH Actin and p Tubulin
23. l plate and in one 10cm dish 2 When cells are approximately 60 to 70 confluent in the 6 well plate add 100ul of the adenoviral stock at a titer of 10 to TO pfu ml to 0 5ml of complete culture medium in a separate tube Aspirate the culture medium from the 6 well plate and then add the diluted virus onto the 293 cells slowly without dislodging the cells Return the plate to 37 C 5 CO incubator for 1 2 hours before adding another 1 5ml of complete medium into the well It will take 3 6 days to observe over 9076 of the cells detached from the plate CPE 3 While adenovirus is being replicated in the 6 well plate subculture the 10cm dish to five 10cm or one 15cm dish which can then be re subcultured to 5X15cm dishes later on When 293 cells reach 60 to 70 confluency in the 10cm or 15cm dishes add 300 400yl of crude viral stock from the previous 6 well plate directly into the 10cm or 15cm culture dishes It will take another 4 5 days before the completion of CPE B Adenovirus Harvest 4 Collect all cells and culture medium into one or more 50ml conical tubes when CPE is complete 5 Freeze thaw the cells and culture medium 3 times to release virus from cells Complete the freezing step using an ethanol dry ice bath and the thawing step using a 37 C water bath 6 Pellet the cell debris by centrifugation at 2 000g for 10 minutes Viral Purification Handbook Page 6 of 19 Protocol 7 Transfer the supernatant to a new
24. rna tant with syringe filter included in the kit For the Giga kit viral supernatant over 50ml should be split up between two syringe filters Pre filtration will de crease viscosity and improve the flow rate of the sample during virus purifica tion steps Note The syringe filter is a low protein binding type to avoid loss of recom binant virus 4 Clamp the syringe column of the PuRetro Purification Unit onto a ring clamp stand Place a beaker under the filter unit for waste collection Alternatively a syringe pump can be used for the following procedures Viral Purification Handbook Page 12 of 19 Protocol 5 Equilibrate PuRetro Purification Unit with 5ml of Equilibration Buffer or a Giga PuRetro Purification Unit with 10ml of Equilibration Buffer Place the feed tube into a 50ml conical tube containing Equilibration Buffer and pull up the syringe plunger to withdraw 5 or 10ml of Buffer Dispense the buffer through the purification unit by pressing down lightly on the syringe plunger The buffer should flow through the filter unit at a rate of 3ml min approxi mately 1 drop per second Note Avoid dispensing air through the PuRetro Purification Unit The pres ence of air in the filter may cause uneven distribution of solutions in the mem brane and alter the efficiency of recombinant retrovirus and lentivirus pu rification Remove the filter first before depleting the residual air from the syringe 6
25. rocess takes less than 2 hours to complete whereas the Cesium chloride method takes 48 hours to complete the same task The Add N Pure purification unit comes pre assembled in the kit for immediate use The one way valve allows selective withdrawal and elution in the desired direction facilitating the purification process The adenovirus purification filter contains an ion exchange membrane that has a high affinity for adenovirus and can be efficiently eluted under our proprietary buffer conditions Our Add N Pure system has been tested to be more reliable and consistent than traditional Cesium chloride ultracentrifugation The Add N Pure adenovirus purification kit can purify and concentrate recombinant adenovirus to a concentration between 10 pfu ml to 10 pfu ml de pending on the amount of viral input whether the Mega or Giga filter is used and whether buffer exchange is performed Titers up to 10 to 10 pfu ml can be obtained with Cat A902 and Cat A905 purification units whereas titers of 10 to 10 pfu ml are achievable with the Cat A910 purification unit However it is important to note that the titers are significantly influenced by the original titer of the recombinant adenovirus input Recombinant adenovirus which expresses proteins or regulatory DNAs toxic to packaging 293 cells will result in notably low viral titer in the original STOCK Viral Purification Handbook Page 4 of 19 Add N Pure Kit Contents
26. rough the Add N Pure Purification Unit at a rate of 3ml min approximately 1 drop per second D Adenovirus Elution 14 15 16 17 18 19 20 Remove the plunger from a sterile 10ml syringe provided in the kit Remove the filter from the Add N Pure Purification Unit Attach the filter to the sterile 10ml syringe Add 3ml of Elution Buffer to the syringe column If the Giga filter purifi cation unit is used or the elution is to undergo buffer exchange for in vivo ap plications or higher titer concentration up to 10ml Elution Buffer can be used to increase the elution efficiency Replace the plunger in the syringe Elute and discard the first 1 0ml of Elution Buffer into a waste container The first 1 0ml is largely comprised of Wash Buffer and should not be kept Leave the rest of the buffer in the syringe column Incubate the filter at room temperature for 5 minutes Rest filter on top of a sterile 15ml conical tube to catch any premature elution Elute at a rate of 1 0ml min approximately 1 drop every 3 seconds into a sterile 15ml conical tube Use the residual air in the syringe to help expel remaining Elution Buffer from the filter The eluted buffer contains the purified adenovirus E Post Purification Notes 21 Filter sterilize viral stock post purification using alow protein binding syringe filter unit provided in the kit Note This is to be done only if Viral Stock Buffer Exchange is
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