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Biomax 100 k concentrator

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The Biomax 100 K concentrator is a laboratory product manufactured by Merck Group. It is a centrifugal device used for concentrating and purifying samples, such as proteins, peptides, or other macromolecules, by retaining particles above a specific molecular weight cutoff on a membrane.

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Hitrap, by Cytiva (3 mentions) Akta fplc system, by Cytiva (2 mentions)

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Biomax 100.000 (3 citations)

5 protocols using biomax 100 k concentrator

1

AAV-mediated PTPN11 gene delivery

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The coding sequence of human PTPN11 with or without the D61G mutation was subcloned into the HindIII – NsiI site of the AAV expression vector pSOFF. The resultant vector expresses mutant PTPN11 under the synthetic CBA promoter (CMV enhancer and chicken beta-actin promoter). Recombinant virus (rAAV5) was purified as previously described34 (link). Briefly, an iodixanol gradient purification was performed followed by an ion exchange chromatography step which results in a 99 % pure vector preparation as judged by silver stained-SDS acrylamide gel fractionation. After the chromatography, the buffer was exchanged and the virus was concentrated in Ringer’s solution using a Biomax 100 K concentrator (Millipore). Vector titers were determined by Real Time PCR. Typical titers were 3.09 × 1012 genome copies/ml. rAAV5-GFP expressing only GFP was used as a control. Virus was infused into two sites per hemisphere (1 μl per injection, AP=−2.5, Lat=+/−2, DV=−1.7; AP=−1.8, Lat=+/−1, DV=−1.6) over 5 min through a 30-gauge Hamilton microsyringe. Viruses (GFP, WT PTPN11 or PTPN11D61G) were randomly assigned for infusion. After completion of infusion, the syringe was left in place for an additional 5 min. All the experiments were done three weeks after the infusion.
Lee Y.S., Ehninger D., Zhou M., Oh J.Y., Kang M., Kwak C., Ryu H.H., Butz D., Araki T., Cai Y., Balaji J., Sano Y., Nam C.I., Kim H.K., Kaang B.K., Burger C., Neel B.G, & Silva A.J. (2014). Mechanism and treatment for the learning and memory deficits associated with mouse models of Noonan syndrome. Nature neuroscience, 17(12), 1736-1743.
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2

Purification and Characterization of rAAV Vectors

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Vector preparations were produced by the plasmid co-transfection method as shown previously (Petrs-Silva et al., 2011 (link)). Briefly, the crude iodixanol fraction with rAAV vectors was further purified and concentrated by column chromatography on a 5-ml HiTrap Q Sepharose column using an AKTA FPLC system (Amersham Biosciences, Piscataway, NJ). The vector was eluted from the column using 215 mM NaCl, pH 8.0, and the vector containing fractions were collected, pooled, concentrated, and buffer exchanged into Alcon BSS with 0.014% Tween 20, using a Biomax 100 K concentrator (Millipore, Billerica, MA). The titer of DNase-resistant vector genomes was measured by real-time PCR relative to a standard. Finally, the purity of the vector was validated by silver-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis, assayed for sterility and lack of endotoxin, and then aliquoted and stored at −80°C. Each vector contained the genome encoding green fluorescent protein (GFP) or human CHIP under the control of a ubiquitous chicken beta-actin (CBA) promoter.
Cabral Miranda F., Adão-Novaes J., Hauswirth W.W., Linden R., Petrs-Silva H, & Chiarini L.B. (2015). CHIP, a carboxy terminus HSP-70 interacting protein, prevents cell death induced by endoplasmic reticulum stress in the central nervous system. Frontiers in Cellular Neuroscience, 8, 438.
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3

AAV-mediated PTPN11 gene delivery

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The coding sequence of human PTPN11 with or without the D61G mutation was subcloned into the HindIII – NsiI site of the AAV expression vector pSOFF. The resultant vector expresses mutant PTPN11 under the synthetic CBA promoter (CMV enhancer and chicken beta-actin promoter). Recombinant virus (rAAV5) was purified as previously described34 (link). Briefly, an iodixanol gradient purification was performed followed by an ion exchange chromatography step which results in a 99 % pure vector preparation as judged by silver stained-SDS acrylamide gel fractionation. After the chromatography, the buffer was exchanged and the virus was concentrated in Ringer’s solution using a Biomax 100 K concentrator (Millipore). Vector titers were determined by Real Time PCR. Typical titers were 3.09 × 1012 genome copies/ml. rAAV5-GFP expressing only GFP was used as a control. Virus was infused into two sites per hemisphere (1 μl per injection, AP=−2.5, Lat=+/−2, DV=−1.7; AP=−1.8, Lat=+/−1, DV=−1.6) over 5 min through a 30-gauge Hamilton microsyringe. Viruses (GFP, WT PTPN11 or PTPN11D61G) were randomly assigned for infusion. After completion of infusion, the syringe was left in place for an additional 5 min. All the experiments were done three weeks after the infusion.
Lee Y.S., Ehninger D., Zhou M., Oh J.Y., Kang M., Kwak C., Ryu H.H., Butz D., Araki T., Cai Y., Balaji J., Sano Y., Nam C.I., Kim H.K., Kaang B.K., Burger C., Neel B.G, & Silva A.J. (2014). Mechanism and treatment for the learning and memory deficits associated with mouse models of Noonan syndrome. Nature neuroscience, 17(12), 1736-1743.
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4

Purification of rAAV2 Vectors for Gene Delivery

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All rAAV2 vector preparations were done by a double transfection protocol as previously described.45 (link) Crude iodixanol fractions of rAAV2 vector were further purified and concentrated by chromatography on a 5-mL HiTrap Q Sepharose column using an AKTA FPLC system (Amersham Biosciences, Piscataway, NJ, USA). Vectors were eluted from the column using 215 mM NaCl, pH 8.0, and the vector-containing fractions collected, pooled, concentrated, and buffer exchanged into Alcon BSS with 0.014% Tween 20, using a Biomax 100 K concentrator (Millipore, Burlington, MA, USA). The titer of DNase-resistant vector genomes was measured by real-time PCR relative to a standard. Finally, the purity of the vector was validated by silver-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis, assayed for sterility and lack of endotoxin, and then aliquoted and stored at −80°C. Vectors contained the sequences encoding either humanized green fluorescent protein (hGFP)46 (link) or human MAX under the control of the ubiquitous citomegalovirus (CMV) enhancer/chicken β-actin (CBA) promoter and simian virus 40 late polyadenylation signal (polyA).
Lani-Louzada R., Marra C., Dias M.S., de Araújo V.G., Abreu C.A., Ribas V.T., Adesse D., Allodi S., Chiodo V., Hauswirth W., Petrs-Silva H, & Linden R. (2022). Neuroprotective Gene Therapy by Overexpression of the Transcription Factor MAX in Rat Models of Glaucomatous Neurodegeneration. Investigative Ophthalmology & Visual Science, 63(2), 5.
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5

Tyrosine-mutant AAV9 Vector Purification

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Surface-exposed tyrosine (Y) residues on AAV9 capsids underwent site-directed mutation to phenylalanine (F) to alter the vector transduction characteristics, as previously described [16] . This site-directed mutation resulted in the generation of tyrosine-mutant Y731F AAV9. Vector preparations were produced using the plasmid co-transfection method [17] . The crude iodixanol fraction, as described, was further purified and concentrated by column chromatography on a 5-ml HiTrap Q Sepharose column using a Pharmacia AKTA FPLC system (Amersham Biosciences, Piscataway, NJ). The vector was eluted from the column using 215 mM NaCl (pH 8.0) and the rAAV peak was collected. Vector-containing fractions were then concentrated and buffer exchanged in Alcon BSS with 0.014% Tween 20, using a Biomax 100K concentrator (Millipore, Billerica, MA). Vector was then titered for DNase-resistant vector genomes by real-time polymerase chain reaction (PCR) relative to a standard. The purity of the vector was validated by silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis, assayed for sterility and lack of endotoxin, and then aliquoted and stored at -80°C.
Martini S.V., Silva A.L., Ferreira D., Rabelo R., Ornellas F.M., Gomes K., Rocco P.R., Petrs-Silva H, & Morales M.M. (2016). Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 39(2).
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