The oligomeric state of FnYggS was assessed by performing size exclusion chromatography with a Superdex 200 10/600 GL column (GE Healthcare, Chicago, IL, USA). A microsyringe was used to inject 500 μL of purified FnYggS (0.5 mg mL−1) into the column. The column was pre-equilibrated with a column buffer consisting of 20 mM HEPES (pH 8.0), 150 mM NaCl, and 2 mM BME, and the absorbance of the protein was monitored at 280 nm. The molecular weights of the eluted samples were calculated based on the calibration curves by several standard samples (Gel Filtration Markers Kit for Protein Molecular Weights 12,000–200,000 Da, Sigma–Aldrich, St. Louis, MO, USA), such as cytochrome c, carbonic anhydrase, albumin, alcohol dehydrogenase, β-amylase, and blue dextran.
Gel filtration markers kit for protein molecular weights 12 000 200 000 da
Manufactured by Merck Group
Sourced in United States
The Gel Filtration Markers Kit for Protein Molecular Weights 12,000–200,000 Da is a laboratory product designed to determine the molecular weights of proteins using gel filtration chromatography. The kit contains a set of protein standards with known molecular weights within the specified range.
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5 protocols using gel filtration markers kit for protein molecular weights 12 000 200 000 da
1
Oligomeric State Analysis of FnYggS
2
Fn1419 Protein Purification by Gel Filtration
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Purified Fn1419 (500 μL; 5 mg/mL) was loaded onto a Superdex 200 10/300 GL column (GE Healthcare, USA), and the protein was eluted using a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2 mM β-mercaptoethanol, at a flow rate of 0.3 mg/mL. The molecular weights of the eluted samples were calculated based on the calibration curves by β-amylase and alcohol dehydrogenase (Gel Filtration Markers Kit for Protein Molecular Weights 12,000–200,000 Da, Sigma-Aldrich, St. Louis, MO, USA).
3
Purification and Characterization of CASP4-nleF Complex
4
Purification and Characterization of CASP4-nleF Complex
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BL21 Star cells were co-transformed with pACYC-DUET-1-CASP4C258S His-p20/p10 and pMAL-c2x-nleFEPEC. Bacterial pellets were re-suspended in 20mM Tris-HCl pH 7.4, 250 mM NaCl and lysed by sonication and purified by amylose affinity chromatography. Bacterial lysates were incubated with amylose resin for 1.5 h at 4 °C and then washed with 50 ml wash buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl and eluted with wash buffer supplemented with 10 mM maltose. The co-elute was dialysed and then purified further by IMAC talon affinity chromatography, as described previously(41 (link)). Complex formation was analysed by size exclusion (Akta prime) with a Superdex200 column (GE Healthcare) using the Gel Filtration Markers Kit for Protein Molecular Weights 12,000-200,000 Da (Sigma-Aldrich) to determine complex size. Size exclusion fractions were verified by SDS PAGE gel and confirmed by Mass spectrometry.
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Gel Filtration Analysis of CarFS.39006 Complex
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