ViraBind™ AAV Purification Mega Kit
Contents
1. Product Manual ViraBind AAV Purification Mega Kit Catalog Number VPK 141 2 preps VPK 141 5 10 preps FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction The viral system includes vectors developed from retrovirus RV adenovirus AdV adeno associated virus AAV lentivirus LV and herpes simplex virus HSV AAV belongs to the family of Parvoviridae a group of viruses among the smallest of single stranded and non enveloped DNA viruses There are eight different AAV serotypes reported to date Recombinant AAV 2 is the most common serotype used in gene delivery and can be produced at high titers with a helper virus or Cell Biolabs AAV Helper Free System AAV 2 can infect both dividing and non dividing cells and can be maintained in the human host cell creating the potential for long term gene transfer Because AAV 2 is a naturally defective virus requiring provision of several factors in trans for productive infection it is considered the safest viral vector to use Recently a new vector AAV DJ was developed using DNA family shuffling to create a hybrid capsid from 8 different AAV serotypes resulting in a vector with significantly higher in vitro infection rates across a variety of cells and tissues Recombinant AAV 2 and AAV DJ vectors can be purified by CsCl gradient ultracentrifugation iodixanol discontinuous gradient ultrace2. 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 8 Ja cree BioLABs NC 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 2007 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing eo CELL BIOLABS INC AN
3. V DJ Samples The following procedure is suggested for 10 x 15 cm dishes and may be optimized to suit individual needs Please refer to the user manual when using Cell Biolabs AAV Helper Free System or other packaging systems 1 Use HEK 293 cells that have been passaged 2 3 times prior to transfection Culture these cells until the monolayer is 70 80 confluent 2 Cotransfect cells with the pAAV RC pHelper and your expression construct according to manufacturer s manual 3 After 48 72 hrs add 0 5 M EDTA to a final of 10 mM to the plate and incubate for 3 min at room temperature Gently shake the culture plate several times and harvest all media including cells in a sterile tube Note Trypsin may be substituted for EDTA in this step 4 Centrifuge for 5 min at 3000 rpm to pellet the transfected cells Resuspend the cell pellet in 2 5 mL of serum free DMEM per 15 cm dish 25 mL total for 10 x 15 cm dishes 5 Subject the cell suspension to four rounds of freeze thaw cycles by alternating the tubes between the dry ice ethanol bath and the 37 C water bath 6 Collect the AAV supernatant by centrifugation at 10 000 x g for 10 minutes Discard the pellet 7 Optional For AAV samples produced with a helper adenovirus inactivate the helper adenovirus by incubation the sample at 56 C for 30 minutes 8 Filter the supernatant through a 0 45 um low protein binding filter The viral supernatant can be stored at 80 C or immediately purif
4. ecovery tube collect the concentrated AAV sample by inverting the sample reservoir into the tube and briefly centrifuging to collect all of the liquid 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 ot ae 100 80 60 GFP 40 20 0 NY S o K x S we S s CA X Figure 1 Purification of AAV2 GFP AAV2 GFP was produced by a helper free system in 293 cells AAV supernatant was subjected to the purification steps Samples from each fraction were used to infect 293 cells GFP positive cells were scored by counting after three days A AAV Supernatant B Flow through C 1 wash D Elution 7 h CELL BIOLABS INC P VP1 86 kDa VP2 72 kDa VP3 62 kDa Figure 2 Electrophoretic Profile of Purified AAV2 GFP References 1 Rabinowitz J and Samulski R J 1998 Curr Opin Biotechnol 9 470 475 2 Summer ford C and Samulski R J 1999 Nat Med 5 587 588 3 Clark K Liu X McGrath J and Johnson P 1999 Hum Gene Ther 10 1031 1039 Recent Product Citation Nazari M et al 2014 AAV2 mediated follistatin overexpression induces ovine primary myoblasts proliferation BMC Biotechnol 14 87 Warranty These products are warranted to perform as described in their
5. ied see purification instructions below Protocol I Purification The following procedure is written for purification and concentration of 25 mL of AAV supernatant For AAV samples that are less than 25 mL add serum free DMEM to the final volume of 25 mL oe SNe Add 0 25 mL of ViraBind AAV Reagent A to 25 mL of viral supernatant mixing well Incubate for 30 minutes at 37 C Centrifuge the AAV supernatant for 15 minutes at 5 000 x g Carefully collect the supernatant and transfer to a new tube Discard the pellet Incubate ViraBind AAV Reagent B for 30 60 minutes at 37 C to ensure Reagent B is dissolved Add 1 25 mL of ViraBind AAV Reagent B to the ViraBind AAV Reagent A pretreated 25 mL of viral supernatant mixing well 6 Incubate for 30 minutes at 37 C Filter the supernatant through a 0 45 um low protein binding filter 8 Resuspend the AAV Purification Matrix by inverting and shaking Add 3 mL of matrix to the filtered supernatant CELL BIOLABS INC 9 Mix the supernatant matrix suspension at 4 C for 30 minutes on an orbital shaker 10 Remove the break off tip from the bottom of the Purification Column by twisting and place into an empty 50 mL conical tube y A y Column 4 Packing Material bd Break off H 4 ip 11 Vortex the supernatant matrix suspension and pour the solution into the Purification Column AAV Purification Matrix will collect inside the column with supe
6. ntrifugation and high performance liquid chromatography HPLC The most popular technique CsCl ultracentrifugation is time consuming process which may result in poor recovery and quality nonviral protein contamination and a high ratio of genome copies versus infectious units ViraBind AAV Purification Mega Kit does not involve ultracentrifugation AAV 2 or AAV DJ is first purified from viral supernatant with a single step column then further purified concentrated with a centrifugal concentrator see Assay Principle below The entire procedure takes about 3 hours Each preparation can handle up to 10 x 150 mm dishes resulting in 100 uL of highly purified AAV 2 Figure 1 or AAV DJ with low ratio of genome copies versus infectious units ViraBind AAV Purification Mega Kit provides an efficient system for quick purification of AAV 2 or AAV DJ with high recovery gt 60 The highly purified and concentrated viruses can be used in primary cell infections and in vivo applications CELL BIOLABS INC a Assay Principle P Pretreatment of Cy AAV CS AAV Lysate with ViraBind Reagent A Purification Column CS y and B i Eo i Centrifugal Concentrator Addition of AAV Purification Matrix Collect the AAV Purification Matrix in the Purification Column i Elute the Purified AAV Concentrate the Purified Fraction N CELL BIOLABS INC Creating Solutions for Life Science Research Related Products toy Mo de
7. rnatant dripping through Rinse the AAV supernatant matrix collection tube with 10 mL of 1X PBS and pour the solution into the Purification Column 12 Once the supernatant has completely flowed through and the column stops dripping wash the collected matrix by add 10 mL of Purification Buffer to the inside the column Repeat the wash once more 13 Transfer the column to a clean empty 50 mL conical tube 14 Elute the purified AAV from the purification matrix by slowly adding 3 mL of Elution Buffer collecting the flowthrough II Final Buffer Exchange and Concentration 1 Assemble a Centrifugal Concentrator unit by inserting the sample reservoir into a recovery tube me g some Reservoir Rae 4 Recovery Tube 2 Apply 0 5 mL of the recovered AAV fraction step 14 above to the sample reservoir of the Centrifugal Concentrator Cap the concentrator and place into a tabletop centrifuge Microfuge Centrifuge for 5 minutes at 2 000 x g As the fraction sample is concentrated top off the concentrator with additional AAV fraction centrifuging between Flow through can be discarded 6 gt CELL BIOLABS INC A A 3 Continue to concentrate the AAV fraction until 100 uL remains in the sample reservoir 4 Add 400 uL of 1X PBS or desired final buffer to the Concentrator and continue to centrifuge until 100 uL remains Repeat step 4 once more 5 Finally concentrate until the desired final volume 6 Using a clean r
8. uly VPK 140 ViraBind AAV Purification Kit AAV 100 293AAV Cell Line VPK 145 QuickTiter AAV Quantitation Kit AAV 200 ViraDuctin AAV Transduction Kit VPK 099 ViraBind Adenovirus Miniprep Kit VPK 100 ViraBind Adenovirus Purification Kit VPK 104 ViraBind Lentivirus Purification Kit Kit Components Sl Oy OW Poe YS ViraBind AAV Reagent A Part No 314001 Two 0 3 mL sterile tubes ViraBind AAV Reagent B Part No 314002 Two 1 5 mL sterile tubes AAV Purification Matrix Part No 314103 One 6 mL bottle Purification Columns Part No 314104 Two empty columns Purification Buffer Part No 314105 One 50 mL bottle Elution Buffer Part No 314106 One 10 mL bottle Centrifugal Concentrators Part No 309505 Pack of 2 units with 4 recovery tubes Materials Not Supplied aA PY yy S AAV Helper Free System Transfection Reagent HEK 293 cells and cell culture growth medium Centrifuge capable of 10 000 x g 0 45 um low protein binding filter 50 mL Conical Tubes Storage Store ViraBind AAV Reagent B Purification Columns and Centrifugal Concentrators at room temperature and all other kit components at 4 C until their expiration dates 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 CELL BIOLABS INC ae Preparation of rAAV 2 or rAA
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