Size-exclusion chromatography (SEC) analysis was performed using a Superdex 75 10/300 GL prepacked column connected to ÄKTA pure 25 chromatography system (GE Healthcare). The column was equilibrated with a 50 mM potassium phosphate buffer (pH 7.0), 150 mM NaCl and then loaded with a 500 μL sample of globupain protein (1 mg/mL). The flow rate of the run was adjusted to 0.5 mL/min, and the absorbance was measured at 280 nm (mAU, milli-absorbance units). For the experiment, the column was calibrated with proteins of known molecular weight: alcohol dehydrogenase (tetramer), 146,800; bovine serum albumin, 66,000; ovalbumin, 43,000; trypsin inhibitor, 22,000; and cytochrome C, 12,400 (Sigma-Aldrich, St. Louis, MO, United States). Dextran blue 2000 (Cytiva) was used to determine the column void volume.
Kta pure 25 chromatography system
Manufactured by GE Healthcare
Sourced in United States
The ÄKTA pure 25 is a chromatography system designed for purification of biomolecules. It is capable of performing a range of chromatographic techniques, including ion exchange, size exclusion, and affinity chromatography. The system is equipped with advanced software and user-friendly interfaces to monitor and control the purification process.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin inhibitor, by Merck Group (2 mentions)
Blue dextran 2000, by Cytiva (2 mentions)
Superdex 75 10 300 gl, by GE Healthcare (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ovalbumin (ova), by Merck Group (2 mentions)
Cytochrome c, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Gel filtration markers kit, by Merck Group (1 mentions)
Blue dextran, by Merck Group (1 mentions)
Potassium ferricyanide, by Fujifilm (1 mentions)
Astra analysis software, by Wyatt Technology (1 mentions)
Dawn 8, by Wyatt Technology (1 mentions)
Optilab t rex, by Wyatt Technology (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Superose 6 increase 10 300 gl, by GE Healthcare (1 mentions)
Ktaprime plus, by GE Healthcare (1 mentions)
Hiscreen capto core 700 column, by GE Healthcare (1 mentions)
Hiscreen, by Cytiva (1 mentions)
Capto, by Cytiva (1 mentions)
7 protocols using kta pure 25 chromatography system
1
Size-Exclusion Chromatography of Globupain
2
Purification and Characterization of PtoMBD Enzyme
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PtoMBD purified with affinity chromatography was buffer exchanged with 20 mM sodium phosphate, pH 7.4, containing 500 mM NaCl and concentrated using a Vivaspin Turbo 4 (10,000 MWCO) ultracentrifugation unit (Merck Millipore). The concentrated enzyme solution was loaded onto a HiLoad 16/600 Superdex 200 prep grade gel-filtration column (GE Healthcare) and eluted with the same buffer at a flow rate of 0.6 ml/min using an ÄKTA pure 25 chromatography system (GE Healthcare). The elution of protein was monitored by UV absorption at 280 nm. A standard curve for calibration was obtained using a gel filtration markers kit (Sigma–Aldrich) containing blue dextran (2000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa) and potassium ferricyanide (Wako).
3
SEC-based Protein Molecular Weight Determination
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SEC analysis was performed using a Superdex 75 10/300 GL prepacked column connected to ÄKTA pure 25 chromatography system (GE Healthcare). The column was equilibrated with a 50 mM potassium phosphate buffer (pH 7.0), 150 mM NaCl and then loaded with a 500 μL sample of globupain protein (1 mg/mL). The flow rate of the run was adjusted to 0.5 mL/min, and the absorbance was measured at 280 nm (mAU, milli-absorbance units). For the experiment, the column was calibrated with proteins of known molecular weight: alcohol dehydrogenase (tetramer), 146,800; bovine serum albumin, 66,000; ovalbumin, 43,000; trypsin inhibitor, 22,000; and cytochrome C, 12,400 (Sigma-Aldrich, St. Louis, MO, USA). Dextran blue 2000 (Cytiva) was used to determine the column void volume.
4
Determination of Galactan Molecular Mass
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An Äkta pure 25 chromatography system (GE Healthcare Bio-Sciences, Marlborough, MA, USA) coupled with a multi-angle light scattering detector (DAWN8+, Wyatt Technology Corporation, Santa Barbara, CA, USA) and an RI-detector (Optilab T-rEX, Wyatt Technology Corporation, Santa Barbara, CA, USA) was used for determination of absolute molecular mass of the galactans. These were dissolved in elution buffer (0.15 M NaCl, 0.05 M phosphate buffer, pH 7.0) and filtered through a sterile filter (Rotilabo PVDF syringe filter, 0.22 µm pore-size, Roth GmbH & CO.KG, Karlsruhe, Germany) prior to injection of 100 µL. The whole system including the Shodex OHpak SB-804 column (Showa Denko AG, Tokyo, Japan) was equilibrated with the same buffer before injection and elution (flow rate was 0.7 mL/min). Molecular weight calculation was performed with the Astra analysis software (v7.1.2, Wyatt Technology Corp.). Pullulan standards (112.0 kDa, 22.8 kDa, 11.8 kDa; PL polysaccharide standard kit SAC-10, Varian Inc., Palo Alto, CA, USA) were investigated likewise and shown in Figure 2 .
5
FPLC Purification of SPEAR Samples
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The prepared SPEAR samples were separated by fast protein liquid chromatography (FPLC) on an ÄKTA pure 25 chromatography system (GE Healthcare) fitted with a Superdex 200 Increase 10/300 GL column (GE Healthcare). In brief, protein samples were first centrifuged at 13,000 ×g for 10 min at 4 °C to remove any precipitants. Then a 500 μl protein sample was loaded onto the column pre-equilibrated with 100 mM carbonate/bicarbonate buffer (pH 10.0) and eluted with the same column buffer at a flow rate of 0.4 mL min−1. The absorbance of the column eluate was continuously monitored at 280 nm. Gaussian fitting on the obtained ultraviolet absorbance tracing over elution volumes was performed manually for peak detection. Molecular weight estimation was performed separately for IgG and Fab due to the non-linear molecular size calibration curve obtained, as well as significant deviations from predicted sizes for proteins reacted with crosslinkers.
6
Analyzing PRA Aggregation via SEC
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The size-exclusion chromatography (SEC) was conducted to analyze the aggregation of wild-type PRA and PRA mutants. Briefly, SuperoseTM 6 increase 10/300GL (GE healthcare) were connected to ÄKTA pure 25 chromatography system (GE healthcare) and balanced using elution buffer (0.1 mol NaCl, 0.05 mol NaH2PO4. 2H2O, pH 7.5) first. Next, 1 mg wild-type PRA and PRA mutants were inputted and eluted using same elution buffer with elution speed of 0.5 mL/min. The absorbances peaks (mAU 280 nm) for wild-type PRA and PRA mutants were monitored and recorded for further analysis.
7
Optimized Extracellular Vesicle Isolation
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EV isolation was based on the recently optimized isolation techniques utilized in our group and described in a recent publication 41 (link) . Briefly, conditioned media (CM) was harvested and spun first at 500 g for 5 minutes to remove cells, followed by 2,000 g for 10 minutes to remove cell debris and thereafter filtrated through an 0.22 μm filter to remove any larger particles. The CM was then run through a hollow fiber filter (D06-E300-05-N, MIDIKROS 65CM 300K MPES 0.5MM, Spectrum Laboratories) using a tangential flow filtration (TFF) system (KR2i TFF System, Spectrum Laboratories) at a flow rate of 100 ml/min (transmembrane pressure at 3.0 psi and shear rate at 3700 sec -1 ) and concentrated down to approx. 40-50 ml after diafiltration of PBS. The pre-concentrated CM was subsequently loaded onto BE-SEC columns (HiScreen Capto Core 700 column, GE Healthcare Life Sciences) and connected to an ÄKTAprime plus or ÄKTA Pure 25 chromatography system (GE Healthcare Life Sciences). Flow rate settings for column equilibration, sample loading and column cleaning in place (CIP) procedure were chosen according to the manufacturer's instructions. The EV sample was collected according to the 280 nm UV absorbance chromatogram and concentrated using an Amicon Ultra-15 10 kDa molecular weight cut-off spin-filter (Millipore).
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