ViraBind™ PLUS Lentivirus Concentration and Purification Mega Kit
Contents
1. LL2. Centrifuge capable of 10 000 x g Storage Store all kit components at 4 C until their expiration dates Preparation of Reagents Dialysis Buffer Prepare sufficient dialysis buffer 25 mM Tris 1 M NaCl pH 7 5 for each purification CELL BIOLABS INC ee e D IREA RKAN 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 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 the lentivirus expression systems from Invitrogen or System Biosciences are 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 2 Replace the cell culture media with new growth media 10 mL per 10 cm dish 3 Transfect cells with the packaging plasmid mix and your expression construct When using 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 Protocol I Purification a
3. Bind Reagent A ED and B to Lentivirus CS Supernatant E Lentivirus WW Centrifugal Concentrator H i JP ont cove Dissolve Virus Complex Pellet and Apply to Dialysis Device Centrifuge to Collect the Lentivirus Pellet Collect and Concentrate the Purified Virus Ja GFE BIOLABS INC DAE NARA AH Related Products a p a VPK 107 QuickTiter Lentivirus Titer Kit Lentivirus Associated HIV p24 VPK 108 HIV p24 QuickTiter Lentivirus Quantitation Kit HIV p24 ELISA VPK 108 QuickTiter Lentivirus Quantitation Kit LTV 200 ViraDuctin Lentivirus Transduction Kit LTV 100 293LTV Cell Line VPK 130 ViraBind Retrovirus Concentration and Purification Kit VPK 100 ViraBind Adenovirus Purification Kit Kit Components l 2 ViraBind Lentivirus Reagent A 100X Part No 309601 One sterile bottle 10 mL ViraBind Lentivirus Reagent B 100X Part No 309602 One sterile bottle 10 mL 3 Dialysis Devices Part No 309603 Two units with 2 floatation rings and disposable recovery pipettes Centrifugal Concentrators Part No 309604 Two units with 2 centrifuge tubes 5 Purification Buffer Part No 309504 One bottle 25 0 mL Materials Not Supplied oN we ES Lentivirus packaging plasmid mix and expression construct Transfection Reagent HEK 293T cells and cell culture growth medium Dialysis Buffer 25 mM Tris 1 M NaCl pH 7 5 PBS
4. DAE VARA Product Manual ViraBind PLUS Lentivirus Concentration and Purification Mega Kit Catalog Number VPK 096 2 preps VPK 096 5 10 preps FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research D IREA RKAN Introduction Lentivirus vectors based on the human immunodeficiency virus 1 HIV 1 have become a promising vector for gene transfer studies The advantageous feature of lentivirus vector is the ability for 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 concentrat
5. ed by ultracentrifuging As a consequence the ultracentrifugation step also concentrates cellular debris membrane fragments and denatured proteins derived from culture media of virus producing cells This unwanted 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 PLUS Lentivirus Concentration and Purification Mega Kit does not involve ultracentrifugation The lentiviruses are first pelleted from viral supernatant with ViraBind reagents patented technology then further purified and concentrated with a dialysis device and concentrator respectively see Assay Principle below Each preparation can handle up to 500 mL of lentiviral supernatant 10 TU mL resulting in 200 300 uL of highly purified lentivirus 10 TU mL ViraBind PLUS Lentivirus Concentration and Purification Mega Kit provides an efficient system for quick lentiviral purification with high recovery gt 60 The highly purified and concentrated viruses can be used in primary cell infections and in vivo applications The system may be adapted to purification of other viral types such as MMLV based retrovirus N AN CELL BIOLABS INC A Assay Principle D TAG ra Addition of Vira
6. embled Centrifugal Concentrator Place into Centrifuge Tube 2 Apply 8 mL of the recovered lentivirus fraction step 8 above to the sample reservoir of the Centrifugal Concentrator Cap the concentrator and place into a centrifuge for 30 minutes at 5 000 x g Flow through will collect in the centrifuge tube 3 If necessary continue to concentrate the lentivirus fraction until 200 300 uL remains in the sample reservoir 4 Collect the concentrated lentivirus sample from the inside of the Centrifugal Concentrator with a pipette 8 a CELL BIOLABS INC TAG ra RA 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 MW MW STD A B c STD A B c 181 8 181 8 Silver Staining 115 5 115 5 Background 82 2 82 2 VSV G 64 2 64 2 48 8 48 8 37 1 37 1 25 9 25 9 p24 19 4 19 4 14 8 14 8 6 6 Figure 1 Electrophoretic Profile of Purified GFP lentivirus 50 mL of GFP lentiviral supernatant was concentrated and purified according to the described Assay Instructions Lentiviral Supernatant A Virus Pellet B and Purified Virus Fraction C were analyzed on SDS PAGE Proteins were visualized by Commassie blue stain left and silver stain right The VSV G envelope protein and p24 coat protein the most abundant proteins in the vector are indicated 9 CELL BIOLABS INC Creating Solut
7. inutes wetting the membrane completely Once wetted do not allow membrane to dry out 3 Remove the dialysis device from the water discarding all liquid from the inside 4 Using a pipette not the included disposable recovery pipette carefully add the lentivirus sample step 6 above to the inside of the dialysis device making sure not to damage the membrane 6 h N CELL BIOLABS INC AN TAG RRAA 5 Screw the blue cap onto the device Carefully push the base of the dialysis device through the hole of the floatation ring in the direction of the blue arrows below Push the floatation ring up until it meets the collar lt Screw on Cap pul Collar Floatation Ring at 3 Floatation Ring 6 Float the dialysis device vertically in a container with a stir bar Dialyze against 4 L of Dialysis Buffer see Preparation of Reagents for 4 6 hours at 4 C with gentle stirring Note Avoid creating a strong vortex which can tip or submerge the device 7 Transfer the dialysis device to another container of PBS or desired buffer Dialyze against 4 L for 4 hours to overnight at 4 C with gentle stirring Repeat this dialysis once more 8 After dialysis is complete open the screw on cap and recover the dialyzed lentivirus sample with the included disposable recovery pipette CELL BIOLABS INC JI gt gt eo TAG rata II Final Concentration 1 Remove the blue cap from the Centrifugal Concentrator unit pre ass
8. ions for Life Science Research D WAG ARAA 1 8 e p24 in supernatant i E p24 in pellet OD 450nm 0 25 50 75 100 p24 ng mL Figure 2 Free p24 does not complex with ViraBind Recombinant p24 diluted in culture medium was treated with ViraBind Lentivirus Reagents The amount of p24 in supernatant and pellet was measured by p24 ELISA Cat VPK 107 Lentivirus Associated HIV p24 ELISA 100 o ez p24 Titer N Supernatant Pellet Purified Virus Figure 3 Vector Yield Determined by p24 ELISA GFP lentiviral supernatant was concentrated and purified according to the Assay Instructions Purification fractions of Lentiviral Supernatant A Virus Pellet B and Purified Virus Fraction C were used to infect 293 cells with GFP expression determined after 72 hr The p24 titer of each fraction was determined by p24 ELISA Cat VPK 107 Lentivirus Associated HIV p24 ELISA 10 a CELL BIOLABS INC D DAE Vr RRAA 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 Notice t
9. nd Concentration The following procedure is written for purification and concentration of 500 mL of lentiviral supernatant For lentiviral samples that are less than 500 mL the amount of ViraBind Lentivirus Reagent A B step 1 and Purification Buffer step 5 needed should be calculated proportionally 1 Add 5 mL of ViraBind Lentivirus Reagent A 100X to 500 mL of viral supernatant mix by inverting Immediately add 5 mL of ViraBind Lentivirus Reagent B 100X and mix by inverting Incubate for 60 minutes at 37 C Centrifuge the complexed lentivirus for 15 minutes at 10 000 x g A pellet should be visible Carefully aspirate the media and collect the pellet ot ae i Resuspend and dissolve the complexed lentivirus pellet in 8 mL of Purification Buffer Vortex the solution to dissolve the pellet Note The solution may appear hazy 6 Centrifuge the solution for 5 minutes at 10 000 x g to remove any insoluble material Carefully recover the supernatant CELL BIOLABS INC A AU e D DAE ARAAT II Dialysis 1 Carefully remove the Dialysis Device from the packaging Firmly holding the collar slowly twist the protective sleeve and pull away from the collar Note Pull the sleeve straight out to avoid damaging the dialysis membrane lt Screw on Cap lt Collar lt Dialysis Device Membrane Protective Sleeve 2 Unscrew the blue cap and carefully submerge the unit in deionized water for 15 30 m
10. o Purchaser This product is sold for research and development purposes only and is not to be incorporated into products for resale without written permission from Cell Biolabs The patented technology is covered by an exclusive license By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses You may contact our Business Development department at busdev cellbiolabs com for information on sublicensing this technology Warranty These products are warranted to perform 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 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 2008 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 11 AN CELL BIOLABS INC
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