The SEC-MALS analysis was performed on a DAWN HELEOS multi-angle light scattering detector, with eighteen angles detectors and a 658.9 nm laser beam, (Wyatt Technology, Santa Barbara, CA, USA) and an Optilab T-rEX refractometer (Wyatt Technology) in-line with two coupled Superdex 200 Increase 10/300 GL size exclusion chromatography analytical columns. Experiments were performed using an isocratic pump (Agilent) with a flow of 0.5 ml/min at room temperature (25°C). Data collection was performed with ASTRA 6.1 software (Wyatt Technology). For the experiments, 300 μl at 1.3 mg/ml protein were loaded on the columns with running buffer of 25 mM HEPES pH 8, 10% glycerol, 300 mM NaCl and 2 mM EDTA. Calibration was performed with high-molecular weight markers from GE Healthcare (thyroglobulin, 669 000 Da; ferritin, 440 000 Da; aldolase, 158 000 Da; conalbumin, 75 000 Da; and ovoalbumin, 43 000 Da).
Ovoalbumin
Manufactured by GE Healthcare
Ovoalbumin is a type of laboratory equipment used in research and diagnostic applications. It is a protein derived from egg white that is commonly used as a standard or reference material in various analytical and biological assays.
Automatically generated - may contain errors
Lab products found in correlation
Thyroglobulin, by GE Healthcare (2 mentions)
Aldolase, by GE Healthcare (2 mentions)
Conalbumin, by GE Healthcare (2 mentions)
Isocratic pump, by Agilent Technologies (1 mentions)
Ferritin, by GE Healthcare (1 mentions)
Dawn heleos multi angle light scattering detector, by Wyatt Technology (1 mentions)
658.9 nm laser beam, by Wyatt Technology (1 mentions)
Astra 6, by Wyatt Technology (1 mentions)
Optilab t rex refractometer, by Wyatt Technology (1 mentions)
Bovine serum albumin (bsa), by GE Healthcare (1 mentions)
Catalase, by GE Healthcare (1 mentions)
Akta explorer, by GE Healthcare (1 mentions)
Chymotrypsinogen a, by GE Healthcare (1 mentions)
Rnase a, by GE Healthcare (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Bovine trypsin, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Ribonuclease, by GE Healthcare (1 mentions)
Blue dextran, by GE Healthcare (1 mentions)
Albumin, by GE Healthcare (1 mentions)
4 protocols using ovoalbumin
1
Protein Characterization by SEC-MALS
2
Protein Purification and Characterization
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Protein (1 mg/ml; 400 ul) was applied to a Superdex 75 analytical column (30 × 1 cm; GE-Healthcare) using AKTA Explorer (GE-Healthcare) and eluted at a flow rate of 0.8 ml/min in buffer PBS monitoring absorbance at 280, 260 and 220 nm. Molecular weight standards catalase (232 kDa), aldolase (163 kDa), BSA (67 kDa), OvoAlbumin (44 kDa), Chymotrypsinogen A (25 kDa) and RNaseA (13.7 kDa) (GE-Healthcare) were used. For CD40-FasL containing medium the molecular weight standards are as presented in Additional file 2 : Figure S2A.
3
Proteolysis Sensitivity of Structured Proteins
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Proteolysis sensitivity was assessed using bovine trypsin (Sigma) in a molecular ratio (protein/trypsin) of 20/1 and 100/1. Conalbumin and Ovoalbumin (GE Life Science) were used as models of well-structured proteins for direct comparison. All reactions were checked by comassie stained 15% SDS-PAGE.
4
Molecular Mass Characterization of Soluble Fractions
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Soluble fractions were analyzed in a Pharmacia LKB (Uppsala, Sweden), FPLC System, using the molecular exclusion column (GE-Healthcare total volume, Vt = 25 mL) Superose-6 N°3 (range: 5-5000 kDa). It was calibrated with blue dextran (void volume, Vo = 7.16 mL), thyroglobulin (669 kDa), alcohol dehydrogenase (150 kDa), albumin (67 kDa), ovoalbumin (44 kDa), and ribonuclease (19 kDa) from GE Healthcare. The molecular masses of the fractions were calculated using the equation:
where MW is the molecular weight in kDa and V e is the elution volume in mL. Soluble fractions were obtained by dissolving the sample in buffer 28 mmol/L Na 2 HPO 4 , 7 mmol/L NaH 2 PO 4 , pH 7.8 and incubating it for 1 h at 25 °C with agitation. Samples were then centrifuged at 10,000×g for 30 min at 15 °C. 200 μL of the soluble fraction were loaded in the column and eluted in the same buffer at 0.2 mL/min. Polypeptides and peptides were detected by absorbance at 280 nm. Every determination was performed at least twice.
Percentage areas of the peaks were calculated by determining the total area of each chromatogram and the area of every peak obtained. These data was processed with the Origin 8.0 program, peak analyzer function.
where MW is the molecular weight in kDa and V e is the elution volume in mL. Soluble fractions were obtained by dissolving the sample in buffer 28 mmol/L Na 2 HPO 4 , 7 mmol/L NaH 2 PO 4 , pH 7.8 and incubating it for 1 h at 25 °C with agitation. Samples were then centrifuged at 10,000×g for 30 min at 15 °C. 200 μL of the soluble fraction were loaded in the column and eluted in the same buffer at 0.2 mL/min. Polypeptides and peptides were detected by absorbance at 280 nm. Every determination was performed at least twice.
Percentage areas of the peaks were calculated by determining the total area of each chromatogram and the area of every peak obtained. These data was processed with the Origin 8.0 program, peak analyzer function.
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