ViraBind™ Lentivirus Purification Kit (10 preps)
Contents
1. 2 Replace the cell culture media with new growth media 10 mL per 10 cm dish 3 Transfect cells with packaging plasmid mix and your expression construct When use Lipofectamine please refer to Invitrogen s Lipofectamine reagent manual 4 After 36 48 hrs harvest all 10 mL medium in a 15 mL conical tube and centrifuge for 5 min at 3000 rpm to pellet the cell debris Filter the supernatant through a 0 45 um low protein binding filter 5 The viral supernatant can be stored at 80 C or immediately purified see purification instructions below Note Freezing and thawing may result in 2 3 fold loss of viral titer after each cycle 3 CELL BIOLABS INC r D Purification Protocol 1 Prior to application of the viral supernatant to the purification filter the supernatant is clarified by passing through a 0 45 um sterile filter Attach a syringe to the purification filter and add 5 mL of 1X LTV Wash Buffer to pre rinse the filter Slowly allow the viral supernatant to pass through the purification filter by gravity flow and save the flow through To ensure maximal recovery pass the flow through through the same filter again Note When the flow through noticeably slows down during loading viral sample or reapplying the first flow through for the second time gently apply pressure with syringe plunger To ensure maximal virus binding try to keep the flow rate at less than 10 mL min we recommend flow through at2. Product Manual ViraBind Lentivirus Purification Kit Catalog Number VPK 104 10 preps FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Lentivirus vectors based on the human immunodeficiency virus 1 HIV 1 have become promising vectors for gene transfer studies The advantageous feature of a lentivirus vector is the ability of gene transfer and integration into dividing and non dividing cells The pseudotyped envelope with vesicular stomatitis virus envelope G VSV G protein broadens the target cell range Lentiviral vectors have been shown to deliver genes to neurons lymphocytes and macrophages cell types that previous retrovirus vectors could not be used Lentiviral vectors have also proven to be effective in transducing brain liver muscle and retina in vivo without toxicity or immune responses Recently the lentivirus system is widely used to integrate siRNA efficiently in a wide variety of cell lines and primary cells both in vitro and in vivo Lentivirus particles are produced from 293T cells through transient transfection of 3 or 4 plasmids that encodes for the components of the virion Viral medium containing viral particles produced by packaging cells within 48 72 hr can be harvested and frozen To obtain a higher titer pseudovirus supernatant can be concentrated by ultracentrifuging As a consequence the ultracentrifugation step
3. however to avoid cross contamination we suggest that when the filter is used for the second time the same pseudovirus is applied Example of Results The following figures demonstrate typical purification results One should use the data below for reference only This data should not be used to interpret actual results CELL BIOLABS INC T 2 S 9 a Oo a a ra 0 Figure 1 Purification of pseudotyped GFP lentivirus GFP lentiviral supernatant was purified according to the above Purification Instructions Each fraction obtained during purification and its dilution were used to infect Hela cells for 48 hr in the presence of 5 ug of Polybrene 5 ug ml GFP positive cells were scored under high magnification fields References 1 Naldini L U Blomer P Gallay D Ory R Mulligan F H Gage I M Verma and D Trono 1996 Science 272 263 267 2 Verma I M and N Somia 1997 Nature 389 239 242 Kafri T U Blomer D A Peterson F H Gage and I M Verma 1997 Nat Genet 17 314 317 4 Beyer W R M Westphal W Ostertag and D von Laer 2002 J Virol 76 1488 1495 Recent Product Citations 1 Zhou C et al 2015 Lhx8 mediated Wnt and TGF pathways in tooth development and regeneration Biomaterials doi 10 1016 j biomaterials 2015 06 004 2 Ying S Y et al 2006 MicroRNA Protocols Chapter 24 Humana Press Warranty These products are warranted to per
4. 2X LTV Elution Buffer Part No 90203 One bottle 50 mL of 50 mM Tris pH 7 5 5 mM Mg gt Cl 2 M NaCl 2 les CELL BIOLABS INC A _ 4 1X Regeneration Solution Part No 90204 One bottle 50 mL Materials Not Supplied Lentivirus packaging plasmid mix and expression construct Transfection Reagent HEK 293T cells and cell culture growth medium Cell culture centrifuge Glycerol 0 45 um filter 10 or 30 mL size syringe with a Luer Lok tip Oa Ye YY S Storage Store all kit components at room temperature Safety Considerations Remember that you will be working with samples containing infectious virus Follow the recommended NIH guidelines for all materials containing BSL 2 organisms Preparation of Reagents e 1X LTV Wash Buffer Prepare a 1X LTV Wash Buffer by diluting the provided 10X stock 1 10 in deionized water Store the diluted solution at room temperature e 1X LTV Elution Buffer Prepare a 1X LTV Elution Buffer by diluting the provided 2X stock 1 2 in deionized water Store the diluted solution at room temperature Pseudovirus Production The following procedure is suggested for a 10cm dish and may be optimized to suit individual needs Please refer to the user manual when a lentivirus expression systems from Invitrogen or System Biosciences is used 1 Use HEK 293T cells that have been passaged 2 3 times prior to transfection Culture these cells until the monolayer is 70 80 confluent
5. also concentrates cellular debris membrane fragments and denatured proteins derived from culture media of virus producing cells This unwarranted material in the crude vector preparation is toxic to target cells especially primary cells and may cause immunogenic reactions in experimental animal models by in vivo vector administration Therefore to reduce undesirable effects and increase gene transfer efficiency the purification of virus vector becomes essential ViraBind Lentivirus Purification Kit does not involve ultracentrifugation instead it is based on the unique properties of Lentivirus envelop proteins The entire procedure takes 30 minutes Each preparation has a capacity up to 2 0 x 10 IFUs ViraBind Lentivirus Purification Kit provides an efficient system for quick lentiviral purification with high recovery gt 90 The system may be adapted to purification of other viral types such as adenovirus and retrovirus Related Products 1 LTV 100 293LTV Lentiviral Cell Line 2 LTV 200 ViraDuctin Lentivirus Transduction Kit 3 VPK 100 ViraBind Adenovirus Purification Kit 4 VPK 107 QuickTiter Lentivirus Titer Kit Lentivirus Associated HIV p24 5 VPK 108 H QuickTiter Lentivirus Quantitation Kit HIV p24 ELISA 6 VPK 112 QuickTiter Lentivirus Quantitation Kit Kit Components 1 LTV Purification Filter Part No 90200 Five filters 2 10X LTV Wash Buffer Part No 90201 One bottle 100 mL 3
6. dropwise 3 5ml nin Thoroughly wash the purification filter with 10 mL of 1X LTV Wash Buffer using gravity flow or gently applying moderate pressure with syringe plunger Repeat the wash step twice using 10 mL of 1X LTV Wash Buffer each wash Position a collection tube under the purification filter add 2 3 mL of 1X LTV Elution Buffer 25 mM Tris pH 7 5 2 5 mM MgCl 1 M NaCl and allow it to pass through gravity flow or moderate pressure Note Air bubbles between syringe and filter will slow down the elution to avoid them by pipetting a few times be careful not stab the membrane filter When apply moderate pressure it s critical to keep the flow rate slow at drop by drop to ensure the maximal yield Add glycerol to a final concentration of 10 to the purified virus or dialyze the viral solution into a desired buffer Aliquot and store the final purified virus solution at 80 C The purification filter used in step 4 can be used twice Upon completion of the purification add 10 mL of 1X LTV Regeneration Solution to the filter followed by 5 mL of 1X LTV Wash Buffer Wrap the regenerated filter in parafilm and store at 4 C until the next use Notes e The purification filter is designed to be used ONLY twice to ensure high virus recovery gt 90 our results show the virus binding capacity drops significantly when used more than two times e The Regeneration Solution should remove any virus or protein remaining on the filter
7. form as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products AN CELL BIOLABS INC JZ i In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2004 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing CELL BIOLABS INC A A
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