SEC analysis was performed basically according to previous reports21 (link),30 (link),31 (link). Molischianum-LH2 proteins were eluted on a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare) with 20 mM Tris buffer containing 0.02% DDM and 150 mM NaCl (pH 8.0) at the flow rate of 0.4 mL/min.
Hiprep 16 60 sephacryl s 300 hr column
Manufactured by GE Healthcare
The HiPrep 16/60 Sephacryl S-300 HR column is a size exclusion chromatography column designed for the separation and purification of biomolecules. The column features a Sephacryl S-300 HR matrix, which provides high resolution and a wide fractionation range for the separation of proteins, peptides, and other macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Size exclusion chromatography calibration marker kit, by Merck Group (3 mentions)
8 hydroxypyrene 1 3 6 trisulfonic acid, by Thermo Fisher Scientific (1 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine dopc, by Avanti Polar Lipids (1 mentions)
Dgs nta, by Avanti Polar Lipids (1 mentions)
Protein standard, by Bio-Rad (1 mentions)
Chymotrypsin, by Promega (1 mentions)
Hitrap q xl column, by GE Healthcare (1 mentions)
Hiprep 16 60 sephacryl s 200 hr column, by GE Healthcare (1 mentions)
Anti his antibody, by Merck Group (1 mentions)
Spin x uf, by Corning (1 mentions)
13 protocols using hiprep 16 60 sephacryl s 300 hr column
1
SEC Purification of Molischianum-LH2
2
Preparation and Characterization of NTA-Functionalized Liposomes
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Unilamellar liposomes (DOPC, 0.8% DGS-NTA, Avanti Polar Lipids) were prepared as previously described5 (link). Specifically, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids) was combined with 0.8% 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTA[Ni]) (Avanti Polar Lipids), dried with N2 and incubated for 1 h in a vacuum desiccator. Lipids were resuspended in 20 mM Tris pH 8, 35 mM 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS), 50 mM p-xylene-bis-pyridinium bromide (DPX) (Thermo Fischer) and subject to 10× freeze–thaw cycles in dry ice and 42 °C water bath, and 10× extrusions using a 200 µm filter. The liposomes were then purified by gel filtration using a Hi Prep 16/60 Sephacryl S-300 HR column (GE Healthcare) in 150 mM NaCl, 20 mM Tris pH 8 buffer. Proteins were added in a ratio of 1:10,000 with liposomes, with a final liposome concentration of ~400 µM, in 150 mM citrate phosphate buffer ranging from pH 4.0 to 7.5, in 0.5 pH intervals. Assays were done in 96-well opaque plates (Corning), and fluorescence was monitored over a 20-min interval, with readings being taken every 30 s (excitation 403 nm, emission 510 nm). Data were normalized to the percentage of total HPTS fluorescence, by adding 0.3% Triton X-100.
3
Gel Filtration and Western Blot Analysis
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The gel filtration was performed on an HPLC system and a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare) at a rate of 0.5 ml/min, and 0.5 ml fractions were collected every minute. Fractions were separated by 8% SDS–PAGE and analyzed by Western blotting using antibodies recognizing CDC or YFP. The protein standards (Bio-Rad, http://www.bio-rad.com/ ) were used to calibrate the column contain five size standards.
4
Isolation and characterization of egg coat proteins
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Egg coats were isolated from the F1 oocytes of laying white leghorn hens; sperm and sperm protease were prepared from ejaculated semen using gentle centrifugation and nitrogen cavitation, respectively44 (link). In total, 100 mg of isolated egg coat (wet weight) was suspended in 1 mL of PBS by ultrasonication31 (link) and then mixed with the sperm protease preparation. The reaction mixture was kept at 39 °C for 20 h and centrifuged at 16,000 x g for 5 min. The supernatant was 0.45-μm filtered and injected into a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare). SEC elution profiles were monitored by measuring absorbance at λ = 280 nm, and both the ~35 kDa and ~21 kDa N-terminal fragments of cZP1 were analysed by mass spectrometry following in-gel chymotryptic peptide digestion with chymotrypsin (Promega)60 (link).
5
Size-exclusion Chromatography Protein Separation
6
Purification of VP6-VP3 Complex
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The complex of VP6 and VP3 was analyzed by use of a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare) in a buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Note that the VP3 protein was easily aggregated in the presence of a low concentration of NaCl (less than 100 mM). The flow rate was 0.5 ml/min, and 3.0-ml fractions were collected and analyzed by SDS-PAGE.
7
Size-Exclusion Chromatography of Cell Lysates
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A HiPrep 16/60 Sephacryl S-300 HR column (GE) was used to perform size-exclusion chromatography according to the manufacturer’s recommendation. Briefly, cell lysates were loaded onto the column and collected at a flow rate of 0.3 mL/min. Then, the sample fractions were analyzed using WB. The standard curve of the elution was plotted against LogMW by using a size-exclusion chromatography calibration marker kit (Sigma) according to the manufacturer’s recommendation.
8
Protein Fractionation and Identification
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For chromatography experiments, stationary phase cultures of strain CYL12640 were harvested and concentrated 100-fold using Amicon Ultra centrifugal filters with a 30 kDa molecular weight cut off. For size exclusion chromatography, this sample was separated using a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare) without dialysis. For ion exchange chromatography, the sample was separated using a HiTrap Q XL column (GE Healthcare) after dialysis against 50 mM Tris, pH 7.6. For parallel sae mutant comparisons, strain CYL12688 was fractionated in the same manner. In-gel trypsin digestion and tandem mass spectrometry for protein identification was performed by the Proteomics core in the Translational Research Institute at the University of Arkansas for Medical Sciences.
9
Estimation of Enzyme Molecular Mass
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The molecular mass for each expressed enzyme was estimated by size exclusion
chromatography as described byMichlmayr
et al. (2013) , using a HiPrep™ 16/60 Sephacryl®
S − 300 HR column (120 mL; GE Healthcare) at a flow
rate of 0.5 mL min−1, with sodium phosphate
50 mM, NaCl 150 mM, pH 7.0. Calibration using molecular
weight standards in the range 20–700 kDa (Sigma-Aldrich,
Saint-Louis, MO, USA), allowed estimation of the theoretical molecular mass from
Compute pI/MW, ExPASy (https://web.expasy.org/compute_pi/ ).
chromatography as described by
et al. (2013)
S − 300 HR column (120 mL; GE Healthcare) at a flow
rate of 0.5 mL min−1, with sodium phosphate
50 mM, NaCl 150 mM, pH 7.0. Calibration using molecular
weight standards in the range 20–700 kDa (Sigma-Aldrich,
Saint-Louis, MO, USA), allowed estimation of the theoretical molecular mass from
Compute pI/MW, ExPASy (
10
Oligomeric State Determination of KlacADA and KlacGDA
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The oligomeric state of KlacADA and KlacGDA was determined by gel filtration chromatography. Dialysed recombinant proteins were concentrated and KlacADA and KlacGDA were further purified by gel exclusion chromatography by using HiPrep 16/60 Sephacryl S-200 HR column and HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare Life Sciences), respectively. Further confirmation of results was done by MALDI-TOF analysis. MALDI mass spectra of KlacADA and KlacGDA was generated by using sinapinic acid as a matrix on a mass spectrometer (Agilent Technologies G4226A, US).
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