The expression and purification of XadA2 was performed using the vector constructed by Caserta et al. [9 (link)]. The protein was induced using 1 mM isopropyl-β-d -thiogalactopyranoside (IPTG) in a 100 mL culture incubated for 2 h at 37 °C. The cells were collected by centrifugation at 4.000×g for 20 min at 8 °C, as previously described [9 (link)]. The expression pattern of XadA2 was checked by SDS-PAGE 12% stained with Coomassie brilliant blue. The protein was purified using an immobilized metal affinity chromatography (IMAC) column packed with 1.0 mL of nickel-nitrilotriacetic acid (Ni-NTA) resin. Bound proteins were eluted with 4 mL of 200 mM imidazole in 50 mM Tris (pH 7.5), 300 mM NaCl. Aliquots were used to estimate the total protein concentration (Bradford assay) and were analyzed by SDS-PAGE. As the samples were eluted in buffer containing imidazole, a reagent that may influence the results of the bioassays, a chemical removal process was performed using the Amicon® Ultra-0.5 filter devices (Millipore, Burlington, MA, USA) column kit following the manufacturer’s recommendations.
Amicon ultra 0.5 filter devices
Manufactured by Merck Group
Sourced in Germany
The Amicon® Ultra-0.5 filter devices are centrifugal filter units designed for rapid concentration and purification of samples. The devices feature a regenerated cellulose membrane with a molecular weight cutoff to facilitate the separation of molecules based on size.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using amicon ultra 0.5 filter devices
1
Overexpression and Purification of XadA2
2
Quantifying Secreted MMP-2 Activity
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In each experimental condition, zymography (secreted activity) was assessed following the protocol outlined by Ben-Yosef et al. [21] (link). Briefly, the supernatant of cultured cells was harvested and centrifuged at 12,000 RPM to remove any cellular debris. After, supernatant (500 µl) was concentrated (20×) by centrifugation (Millipore -Amicon ultra 0.5 filter devices) and loaded onto Tris-Gly gelatin-containing gels. A 10 ng pro-MMP-2 (70-72 kDa; Abcam; Cambridge, MA, USA) and fully activated MMP-2 (58-62 kDa) standard was loaded on each gel for the evaluation of MMP-2 activity. Densitometric analyses of zymography was completed using the ImageJ software (NIH) to quantify MMP-2 activity in digested zones. Data are expressed as relative band intensity of active MMP-2to-Pro-MMP-2 (mean ± SEM). Representative zymograms blots are shown (n = 4, performed in duplicate).
3
Purification of Adeno-Associated Viruses
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Harvested supernatants and cell pellets were stored at −80 °C and purified using an ÄKTA avant 150 FPLC system with a 5 mL POROS™ GoPure™ AAVX pre-packed column (0.8 × 10 cm) from Thermo Fisher Scientific (Paisley, UK). Samples were loaded at a flow rate of 1 mL/min, followed by a high salt wash step with 50 mM Tris, 1 M NaCl, 2 mM MgCl2 and 0.01% Pluronic at pH 7.5 for 10 column volumes (CVs) and a low pH wash step with 50 mM Tris, 0.2 M NaCl, 2 mM MgCl2 and 0.01% Pluronic at pH 4.0 for 10 CVs. The pH was equilibrated during 10 CVs using 50 mM Tris, 0.2 M NaCl, 2 mM MgCl2 and 0.01% Pluronic at pH 7.5 prior to the elution of the AAVs with 80% 100 mM glycine, 2 mM MgCl2 and 0.01% Pluronic at pH 2.0 for 10 CVs. The eluate was neutralized with 1.0 M Tris and then buffer-exchanged and concentrated in 0.01% Pluronic in 1× PBS solution using 100 kDa Amicon® Ultra-0.5 filter devices (Merck, Darmstadt, Germany).
4
Robust Quantification of rAAV Capsid Composition
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Prior to liquid chromatography separation of empty and full rAAVs, samples were buffer-exchanged into aqueous ammonium acetate (100 mM, pH 6.8) using 100 kDa Amicon® Ultra-0.5 filter devices (Merck, Darmstadt, Germany). Separation was performed using a ProPac SAX-10, 1 × 50 mm column (Thermo Fisher Scientific, Sunnyvale, CA, USA) using FLR detection (Ex280/Em340) on a Vanquish Horizon UHPLC system (Thermo Scientific, Germering, Germany). Buffer A was 20 mM ammonium bicarbonate and 15 mM ammonium hydroxide, pH 10, and buffer B was 15 mM formic acid and 30 mM acetic acid, pH 2.3. At a flow rate of 0.15 mL/min and column temperature of 30 °C, empty and full capsids were separated using the following conditions: samples were loaded onto the column at 0.1% B followed by an increase to 20% B over 1 min. Separation was carried out using a two-step gradient of 20–60% B over 4 min followed by a hold at 60% B for 2.9 min and a second ramp from 60 to 80% B over 8 min. A wash step at 90% B was carried out for 1.5 min and column re-equilibration at 0.1% B for 17.4 min. FLR-detected peak areas were used for calculating the percentage of empty and full capsids in triplicates. One way ANOVA and Tukey’s multiple comparison tests were carried out using GraphPad Prism.
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