ViraBind™ Adenovirus Purification Kit, Trial Size
Contents
1. Product Manual ViraBind Adenovirus Purification Kit Trial Size Catalog Number VPK 100 T 4 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 is2. 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 2013 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing i CELL BIOLABS INC A A
3. both tedious 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 099 ViraBind Adenovirus Miniprep Kit VPK 109 QuickTiter Adenovirus Titer Immunoassay Kit VPK 110 QuickTiter Adenovirus Titer ELISA Kit VPK 111 Rapid RCA Assay Kit VPK 252 RAPAd CMV Adenoviral Expression System VPK 254 RAPAd CMV Adenoviral Bicistronic Expression System GFP So ee Ye YS Kit Components 1 Purification Filter Part No 90001 T Two filters 2 10X Wash Buffer Part No 90002 T One 30 mL bottle 3 2X Elution Buffer Part No 90003 T One 15 mL bottle 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 90004 T One 25 mL bottle Note Each filter is regenerated one time after initial use allowing each filter t
4. cell cell contact 3 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 3 JaN CELL BIOLABS INC A a 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 passing through a 0 45 um sterile filter Attach a syringe to the puri
5. Biotechnol Lett doi 10 1007 s10529 014 1746 4 Fang J et al 2014 PAX6 downregulates miR 124 expression to promote cell migration during embryonic stem cell differentiation Stem Cells Dev 23 2297 2310 Yang M et al 2014 Over expression of JAZF1 reduces proinflammatory cytokines release via inhibition of stress kinases and NF KB FEBS J doi 10 1111 febs 12853 6 CELL BIOLABS INC A a 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Cheng L et al 2014 Induction of specific humoral and cellular immune responses in a mouse model following gene fusion of HSP70C and Hantaan virus Gn and SO 7 in an adenoviral vector PLoS One 9 e88183 Cyphert H A et al 2014 Glucagon stimulates hepatic FGF21 secretion through a PKA and EPAC dependent posttranscriptional mechanism PLoS One 9 e94996 Obokata H et al 2014 Stimulus triggered fate conversion of somatic cells into pluripotency Nature 505 641 647 Haidari M et al 2014 Disruption of endothelial adherens junctions by high glucose is mediated by protein kinase C B dependent vascular endothelial cadherin tyrosine phosphorylation Cardiovasc Diabetol 13 112 Wu D et al 2014 Hypothalamic nesfatin 1 NUCB2 knockdown augments hepatic gluconeogenesis that is correlated with inhibition of mTOR STAT3 signaling pathway in rats Diabetes 63 1234 1247 Araki K et al 2013 Cyt
6. c effects of a double stranded RNA mimic complexed with polycations in an experimental mouse model of endometriosis Fertil Steril doi 10 1016 j fertnstert 2015 07 1147 Miklavc P et al 2015 Actin depolymerisation and crosslinking join forces with myosin II to contract actin coats on fused secretory vesicles J Cell Sci 128 1193 1203 Nuche Berenguer B et al 2015 Elucidation of the roles of the src kinases in pancreatic acinar cell signaling J Cell Biochem 116 22 36 Westermeier F et al 2015 Insulin requires normal expression and signaling of insulin receptor a to reverse gestational diabetes reduced adenosine transport in human umbilical vein endothelium FASEB J 29 37 49 Lakshmanan J et al 2015 Glycogen synthase kinase 3 regulates IL 1 mediated iNOS expression in hepatocytes by down regulating c Jun J Cell Biochem 116 133 141 Brock S E et al 2014 MIF family members cooperatively inhibit p53 expression and activity PLoS One 9 e99795 Kim S M et al 2014 A recombinant adenovirus bicistronically expressing porcine interferon a and interferon y enhances antiviral effects against foot and mouth disease virus Antiviral Res 104 52 58 Zhang J et al 2014 Propofol exerts anti hepatocellular carcinoma by microvesicle mediated transfer of miR 142 3p from macrophage to cancer cells J Transl Med 12 279 Sanchez Lugo Y E et al 2014 CXCL10 XCL1 fusokine elicits in vitro and in vivo chemotaxis
7. factor or for its de encryption Blood 115 4273 4283 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 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 Li H et al 2009 Nuclear translocation of Ad EYFP hCAR a novel tool for screening human CAR activators in human primary hepatocytes Drug Metab Dispos 37 1098 1106 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 Gosselin K et al 2009 Senescence associated oxidative DNA damage promotes the generation of neoplastic cells Cancer Res 69 7917 7925 7 les CELL BIOLABS INC A a 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
8. fication 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 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 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 Before infec
9. o be used twice Materials Not Supplied 1 Recombinant adenovirus of interest 2 HEK 293 cells and cell culture growth medium 3 Cell culture centrifuge 4 Glycerol 5 0 45 um filter 6 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 e 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 e 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 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 2 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 effects CPEs within 48 hrs CPEs on adenovirus infected 293 cells consist of cell rounding swelling loss of
10. oplasmic translocation of the retinoblastoma protein disrupts sarcomeric organization eLife Sci 2 e01228 Scallan C et al 2013 An Adenovirus based vaccine with a double stranded RNA adjuvant protects mice and ferrets against H5N1 avian influenza in oral delivery models Clin Vaccine Immunol 20 85 94 Haidar M et al 2012 Integrin 281 mediates tyrosine phosphorylation of vascular endothelial cadherin induced by invasive breast cancer Cells J Biol Chem 287 32981 32992 Chen F et al 2011 Dynamic regulation of PDX 1 and FoxOl expression 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 AGPAT5S 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 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 Kothari H el at 2010 Cystine 186 cystine 209 disulfide bond is not essential for the procoagulant activity of tissue
11. s 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 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 5 J cete BioLaBs Inc CAM chick chorioallanoic membrane After three days the blood vessels on the CAM were examined under a stereomicroscope References 1 2 3 Stewart P L Fuller S D and Burnett R M 1993 EMBO J 12 2589 2599 Robbins P D Tahara H and Ghivizzani S C 1998 Trends Biotechnol 16 35 40 Huang S Stupack D Mathias P Wang Y and Nemerow G 1997 Proc Natl Acad Sci U S A 94 8156 8161 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 Von Seggern D J C Y Chiu S K Fleck P L Stewart and G R Nemerow 1999 J Virol 73 1601 1608 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 10 Garc a Pascual C M et al 2015 Evaluation of the potential therapeuti
12. t 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 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 i CELL BIOLABS INC 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 amp a gt 60 2 T g 40 20 0 N lt SS N So o X Re a we we Pl S SC Se gt S y X no os GY a Kw oxo Rs R Figure 1 Purification of recombinant Ad B Gal Recombinant Ad B Gal viruses were purified according to the above Purification Instructions Each fraction obtained during purification and it
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