The size exclusion chromatography of xylose-BCH MRPs and BCH samples were performed by using a TSK gel G 2000 SW (600 × 7.5 mm,10 μm, Tosoh Corporation, Tokyo, Japan) equipped with TSK guard column (75 × 7.5 mm, 10 μm). The eluent was 30% acetonitrile solution containing 0.1% TFA distilled water and the flow rate is 0.5 mL/min. First, 20-μL samples (2.5 g/L) were injected, and their absorbances were measured at 214 nm. Bovine serum albumin (66,409 Da), ovalbumin (44,300 Da), trypsin inhibitor (20,100 Da), β-lactoglobulin (36,000 Da), lysozyme (14,300 Da), α-lactalbumin (14,147 Da), oxidized glutathione (612.63 Da), and reduced glutathione (307.32 Da) were adopted as molecular weight (MW).
Tskgel g2000sw
Manufactured by Tosoh
Sourced in Japan
The TSKgel G2000SW is a size exclusion chromatography column used for the separation and analysis of macromolecules, such as proteins, polymers, and other high molecular weight compounds. The column is designed to provide efficient and reliable separation based on the size and molecular weight of the analytes.
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4 protocols using tskgel g2000sw
1
Molecular Weight Determination by SEC
2
Purification and Analysis of NPYs
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The GFC column of TSKgel G2000SW (7.5 × 600 mm; 10 μm) with a guard column (TOSOH Corporation, Tokyo, Japan) was used as the first column. The eluent was an aqueous solution of 40% acetonitrile/0.1%TFA, and a flow rate of 1 ml/min was used. The sample fractions were collected at intervals of 1 min. The lyophilized peptide extract (2.9 mg) was dissolved in 100 μl of 40% acetonitrile/0.1%TFA aqueous solution, and all of them were loaded to the GFC column. The eluted fractions were taken to each tube systematically for 1 min. The NPYs were eluted at the 18 min fraction as shown in Figure 4 , which was dried up by a centrifugal evaporator system (EYELA Tokyo Rikakikai Co. Ltd., Tokyo, Japan), and it was dissolved in 100 μl of 40% acetonitrile/0.1%TFA aqueous solution for the subsequent triple analytical GFC.
3
RRM Protein Size Analysis
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RRM proteins (2, 1, and 0.5 g/L) in 10 mM Na-Pi/100 mM NaCl, pH 7.0 were loaded onto a gel filtration column (TSKgel G2000SW, TOSOH) fitted to the HPLC system (Shimadzu), and the absorbance changes at 280 nm was monitored. A molecular size of the protein eluted from the column was also determined by multi-angle light scattering using miniDAWN TREOS (WYATT Technology) connected on-line to the HPLC system.
4
Purification and Analysis of Plasmodium Chitinases
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Approximately 50 ml of P. gallinaceum ookinete culture supernatant was concentrated using a 10 kDa cut off Centricon device, EMD Millipore, MT to 1 ml final volume. Chitinase activity of the concentrated sample was tested before loading onto a silica-based SEC column (TSKgel G2000SW, Tosoh Biosciences LLC, King of Prussia, PA), and HPLC was performed using a Beckman Gold 500 instrument and autoclaved PBS (pH 7.4). Approximately 100 µl of each fraction (total 0.5 ml volume/1 min) was analyzed in duplicate for the presence of chitinase activity that showed two distinct activity peaks. Gel filtration standards (Bio-rad, Hercules, CA) were analyzed in similar conditions and the retention times of the protein sizes were determined. Similarly, concentrated serum-free ookinete culture supernatant of P. falciparum was subjected to HPLC following the condition for P. gallinaceum; however, it showed low activity in the fractions due to the sample dilutions, but showed two distinct peaks when fractions were coated on ELISA plated and probed with 1C3, anti-PfCHT1 monoclonal antibodies (data not shown). To further resolve the HMW complex and determine the approximate mass, subjected samples were used for high-resolution chromatography, immunodetection, and estimation of the chitinase complex in both P. falciparum and P. gallinaceum.
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