ViraBind™ Adenovirus Purification Kit
Contents
1. Product Manual ViraBind Adenovirus Purification Kit Catalog Number VPK 100 10 preps FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Recombinant adenoviruses have tremendous potential in both research and therapeutic applications There are numerous advantages in using an adenovirus to introduce genetic material into host cells The permissive host cell range is very wide The virus has been used to infect many mammalian cell types both replicative and non replicative for high expression of the recombinant protein Recombinant adenoviruses are especially useful for gene transfer and protein expression in cell lines that have low transfection efficiency with liposome After entering cells the virus remains epichromosomal i e does not integrate into the host chromosome so does not activate or inactivate host genes Recently recombinant adenoviruses have been used to deliver RNAi into cells HEK 293 cells or their variants are used as host cells for viral amplification Recombinant adenoviruses can be grown at high titer 10 VP viral particles mL which can be concentrated up to 10 VP mL The concentrated viral supernatant is subjected to CsCl ultracentrifugation to separate the viruses from the cellular proteins and media components Following ultracentrifugation CsCl is then removed by dialysis The CsCl procedure is both tedious2. e
3. solution into a desired buffer Aliquot and store the final purified virus solution at 80 C Before infect target cells we recommend adding the needed amount of purified virus to 5 10 mL culture medium of your target cells and filter through a 0 2 um sterile filter before infection 6 The purification filter used in step 4 can be used twice Upon completion of the purification add 10 mL of 1X Regeneration Solution to the filter followed by 5 mL of 1X 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 however to avoid cross contamination we suggest that when the filter is used for the second time the same recombinant adenovirus 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 100 80 60 Relative VP 40 20 XN eS amp N we er g Figure 1 Purification of recombinant Ad B Gal Recombinant Ad f Gal viruses were purified according to the above Purification Instructions Each fraction obtained during purifi
4. M Mg gt Cl 2 M NaCl 1X Regeneration Solution Part No 90004 One bottle 50 mL Materials Not Supplied Do po PY YS Recombinant adenovirus of interest HEK 293 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 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 1X Wash Buffer Prepare a 1X Wash Buffer by diluting the provided 10X stock 1 10 in deionized water Store the diluted solution at room temperature 1X Elution Buffer Prepare a 1X Elution Buffer by diluting the provided 2X stock 1 2 in deionized water Store the diluted solution at room temperature CELL BIOLABS INC ae Harvesting Infected Cell Lysate The following procedure is suggested for T75 flasks use only up to 4 x T75 flasks for each purification prep 1 Use HEK 293 cells that have been passaged regularly 2 3 times prior to the infection Culture these cells until the monolayer is 90 confluent Replace the cell culture media with new growth media 15 mL per flask Next add either crude or purified viral stock to the culture Use a multiplicity of gt 0 5 PFU plaque forming units or enough viruses that cells demonstrate cytopathic effe
5. and time consuming 16 24 hrs ViraBind Adenoviral Purification Kit does not involve ultracentrifugation instead it s based on the unique properties of adenoviral capsid proteins The entire procedure takes about 30 minutes Each preparation has a capacity up to 2 5 x 10 VPs ViraBind Adenovirus Purification Kit provides an efficient system for quick adenoviral purification with high recovery gt 90 The system may be adapted to purification of other viral types such as retrovirus and lentivirus The optimal purification conditions for retrovirus are currently under investigation Related Products AD 100 293AD Cell Line AD 200 ViraDuctin Adenovirus Transduction Reagent VPK 090 ViraBind Lentivirus Concentration and Purification Kit VPK 099 ViraBind Adenovirus Miniprep Kit VPK 101 ViraBind Adenovirus Purification Mega Kit VPK 109 QuickTiter Adenovirus Titer Immunoassay Kit VPK 110 QuickTiter Adenovirus Titer ELISA Kit VPK 111 Rapid RCA Assay Kit VPK 130 ViraBind Retrovirus Concentration and Purification Kit 10 VPK 252 RAPAd CMV Adenoviral Expression System 11 VPK 254 RAPAd CMV Adenoviral Bicistronic Expression System GFP oe ee ee S E CELL BIOLABS INC me Kit Components 1 2 3 Purification Filter Part No 90001 Five filters 10X Wash Buffer Part No 90002 One bottle 100 mL 2X Elution Buffer Part No 90003 One bottle 50 mL of 50 mM Tris pH 7 5 5 m
6. ators in human primary hepatocytes Drug Metab Dispos 37 1098 1106 10 Meng Z et al 2009 Forkhead box O1 pancreatic and duodenal homeobox 1 intracellular translocation is regulated by c Jun N terminal kinase and involved in prostaglandin E2 induced pancreatic B cell dysfunction Endocrinology 10 1210 en 2009 0671 11 Gosselin K et al 2009 Senescence associated oxidative DNA damage promotes the generation of neoplastic cells Cancer Res 69 7917 7925 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 2004 2011 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing CELL BIOLABS INC
7. cation and its dilution were used to infect HUVEC cells in a 24 well plate After 48 hrs X gal staining was performed and Gal positive cells blue were scored 5 J CELL BIOLABS INC ae A B Ad Null AD VEGF Figure 2 A X gal staining of infected HUVEC cells HUVEC cells were infected with purified Ad B Gal at 50 MOI multiplicity of infection X gal staining was performed after 48 hr infection period B VEGF induced Angiogenesis Purified Ad Null or Ad VEGF viruses were applied to a 10 day old CAM chick chorioallanoic membrane After three days the blood vessels on the CAM were examined under a stereomicroscope References 1 Stewart P L Fuller S D and Burnett R M 1993 EMBO J 12 2589 2599 2 Robbins P D Tahara H and Ghivizzani S C 1998 Trends Biotechnol 16 35 40 3 Huang S Stupack D Mathias P Wang Y and Nemerow G 1997 Proc Natl Acad Sci U S A 94 8156 8161 4 Bergelson J M J A Cunningham G Droguett E A Kurt Jones A Krithivas J S Hong M S Horwitz R L Crowell and R W Finberg 1997 Science 275 1320 1323 5 Von Seggern D J C Y Chiu S K Fleck P L Stewart and G R Nemerow 1999 J Virol 73 1601 1608 6 Kleinerman D I W W Zhang S H Lin N T Van A C von Eschenbach and J T Hsieh 1995 Cancer Res 55 2831 2836 Recent Product Citations 1 Chen F et al 2011 Dynamic Regulation of PDX 1 and FoxO1 Ex
8. cts CPEs within 48 hrs CPEs on adenovirus infected 293 cells consist of cell rounding swelling loss of cell cell contact During 24 48 hr infection examine the monolayer twice per day under the microscope for CPE When CPE is nearly complete i e most cells rounded but not yet detached from the flask harvest cells by pipetting media up and down to wash the infected cells from the flask into the media Note To achieve the maximal yield for the purification steps it is critical to harvest the infected 293 cells when most of the cells show cytopathic effects but not yet detached from the flask Pool infected cells and medium Pellet cells by centrifugation at 1000 g for 5 minutes Remove all but 15 mL supernatant Note When CPE is nearly complete most viruses are still inside the infected cells before freeze and thaw cycles After centrifugation the excess low titer supernatant may be stored at 80 C for future infection Release the adenoviruses from the cell suspension with three freeze thaw cycles Centrifuge at 3000 g for 10 minutes to pellet the cell debris Discard the pellet and save supernatant If a large amount of cell debris is still visible centrifuge the supernatant again The viral supernatant can be stored at 80 C or immediately purified see purification instructions below Purification Protocol 1 Prior to application of the viral supernatant to the purification filter the supernatant is clarified by pass
9. ing through a 0 45 um sterile filter Attach a syringe to the purification filter and add 5 mL of 1X 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 at dropwise 3 5ml min Thoroughly wash the purification filter with 10 mL of 1X Wash Buffer using gravity flow or gently applying moderate pressure with syringe plunger Repeat the wash step twice using 10 mL of 1X Wash Buffer each wash CELL BIOLABS INC yA A 4 Position a collection tube under the purification filter add 2 3 mL of 1X Elution Buffer 25 mM Tris pH 7 5 2 5 mM Mg gt Cl 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 5 Add glycerol to a final concentration of 10 to the purified virus or dialyze the viral
10. pression by FoxA2 in Dexamethasone Induced Pancreatic B cells Dysfunction Endocrinology 152 1779 1788 Prasad S S et al 2011 Enzymatic Activities of the Human AGPAT Isoform 3 and Isoform 5 Localization of AGPATS to Mitochondria J Lipid Res 52 451 462 Triulzi C et al 2010 Antibody Dependent Natural Killer Cell Mediated Cytotoxicity Engendered by a Kinase Inactive Human HER2 Adenovirus Based Vaccination Mediates Resistance to Breast Tumors Cancer Res 70 7431 7441 Lam Y W et al 2010 Proteomics analysis of the nucleolus in adenovirus infected cells Mol Cell Proteomics 9 117 130 i A CELL BIOLABS INC 5 Sabbatini M E et al 2010 CCK activates RhoA and Racl differentially through G alpha 13 and G alpha q in mouse pancreatic acini Am J Physiol Cell Physiol 298 C592 C605 6 Kothari H el at 2010 Cystine 186 cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de encryption Blood 115 4273 4283 7 Sen P et al 2010 Zinc Modulates the Interaction of Protein C and Activated Protein C with Endothelial Cell Protein C Receptor J Biol Chem 285 20410 20420 8 Agarwal A K et al 2010 Enzymatic activity of the human 1 acylglycerol 3 phosphate O acyltransferase isoform 11 upregulated in breast and cervical cancers J Lipid Res 51 2143 2152 9 Li H et al 2009 Nuclear translocation of Ad EYFP hCAR a novel tool for screening human CAR activ
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