Recombinant FbpC-C, FbpC-C R248A, and FbpC-C H252A proteins were expressed and purified as in (20 (link)). Briefly, after elution of the Ni column using the Elution buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 500 mM Imidazole), the proteins were exchanged into 20 mM Tris (pH 8.0), 500 mM NaCl, 10 mM Imidazole using a Desalting 26/10 column (GE Healthcare). The His-tag was then removed by incubation with the tobacco etch virus (TEV) protease and 5 mM β-mercaptoethanol overnight at room temperature. The TEV digested proteins were separated from the cleaved His-tag product on an AKTA Pure 25L FPLC using a 5 mL HisTrap-FF with the captured flowthrough further purified using a HiLoad Superdex 75 PG gel filtration column (GE Healthcare). A monodisperse peak was obtained and assessed for purity using SDS-PAGE gel analysis, then pooled, concentrated, and exchanged into HBS buffer (10 mM HEPES [pH 7.3], 140 mM NaCl). Purified full length zymogen and active forms of C1r were obtained from Complement Technology, Inc. (Tyler, TX).
Desalting 26 10 column
Manufactured by GE Healthcare
The Desalting 26/10 column is a laboratory equipment designed for the removal of salts and small molecules from protein samples. It features a column size of 26 mm in diameter and 10 cm in length, and is suitable for the rapid desalting of sample volumes up to 2.5 mL.
Automatically generated - may contain errors
Lab products found in correlation
Hiload superdex 75 pg gel filtration column, by GE Healthcare (2 mentions)
Active forms of c1r, by Complement Technology (2 mentions)
Akta pure, by Cytiva (2 mentions)
Amicon ultra 15 concentrator, by Merck Group (2 mentions)
Prism 8, by GraphPad (1 mentions)
Origin 9.1 pro, by OriginLab (1 mentions)
4 protocols using desalting 26 10 column
1
Purification of FbpC-C Proteins
2
Characterization of Apoenzyme Binding
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Apoenzyme was obtained by incubating 5 μΜ holoenzyme with 15 mM hydroxylamine in 0.5 M potassium phosphate buffer, pH 6.8, at room temperature overnight and loaded on a Desalting 26/10 column (GE Healthcare) preequilibrated with 0.5 M potassium phosphate buffer, pH 6.8, and eluted at 1 mL/min. The eluted enzyme was then concentrated on an Amicon Ultra 15 concentrators (Millipore) and washed with 100 mM potassium phosphate buffer, pH 7.4. The equilibrium apparent dissociation constant for PLP, KD(PLP), was determined as described in [20 (link)].
The data were fitted to the following Eq. (1 ):
where [E]t and [PLP]t are the total concentrations of the enzyme and PLP, respectively, Y refers to the intrinsic quenching changes at a PLP concentration, and Ymax refers to the fluorescence changes when all enzyme molecules are complexed with coenzyme. Curves fitting was performed using Prism, 8.4.0 (GraphPad), with automatic confidence interval set at 95% and the values obtained are the mean (± standard error of the mean (SEM)) of three independent experiments.
The data were fitted to the following Eq. (
where [E]t and [PLP]t are the total concentrations of the enzyme and PLP, respectively, Y refers to the intrinsic quenching changes at a PLP concentration, and Ymax refers to the fluorescence changes when all enzyme molecules are complexed with coenzyme. Curves fitting was performed using Prism, 8.4.0 (GraphPad), with automatic confidence interval set at 95% and the values obtained are the mean (± standard error of the mean (SEM)) of three independent experiments.
3
Purification and Characterization of FbpC-C Proteins
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Recombinant FbpC-C, FbpC-C R248A, and FbpC-C H252A proteins were expressed and purified as in (20 ). Briefly, after elution of the Ni column using the elution buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 500 mM imidazole), the proteins were exchanged into 20 mM Tris (pH 8.0), 500 mM NaCl, 10 mM Imidazole using a Desalting 26/10 column (GE Healthcare). The His-tag was then removed by incubation with the tobacco etch virus protease and 5 mM β-mercaptoethanol overnight at room temperature. The tobacco etch virus–digested proteins were separated from the cleaved His-tag product on an AKTA Pure 25L FPLC using a 5 ml HisTrap-FF with the captured flowthrough further purified using a HiLoad Superdex 75 PG gel filtration column (GE Healthcare). A monodisperse peak was obtained and assessed for purity using SDS-PAGE gel analysis, then pooled, concentrated, and exchanged into HBS buffer (10 mM Hepes [pH 7.3], 140 mM NaCl). Purified full-length zymogen and active forms of C1r were obtained from Complement Technology, Inc. Multiple sequence alignments were generated using Clustal Omega (74 (link)).
4
Determination of AADC Apoenzyme KD(PLP)
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Apoenzyme was obtained by incubating 5 µ holoenzyme with 10 mM hydroxylamine in 0.5 M potassium phosphate buffer pH 6.8 at 25°C for 3 hours. The solution was then loaded on a Desalting 26/10 column (GE Healthcare) pre-equilibrated with 0.5 M potassium phosphate buffer pH 6.8 and eluted at 1 mL/min. The eluted enzyme was then concentrated on an Amicon Ultra 15 concentrators (Millipore) and washed with 100 mM potassium phosphate buffer pH 7.4.
The equilibrium apparent dissociation constant for PLP, KD(PLP), was determined by measuring the quenching of the intrinsic fluorescence of 0.1 µM AADC apoenzyme incubated in the presence of PLP at concentrations ranging from 0.005 to 20 μM for 3h at 25°C (in the dark) in 100 mM potassium phosphate buffer pH 7.4.
The data were fitted to the following equation:
where [E]t and [PLP]t represent the total concentrations of the enzyme and PLP, respectively, Y refers to the intrinsic quenching changes at a PLP concentration, and Ymax refers to the fluorescence changes when all enzyme molecules are complexed with coenzyme. Curves fitting was performed using Origin® 9.1 Pro (OriginLab).
The equilibrium apparent dissociation constant for PLP, KD(PLP), was determined by measuring the quenching of the intrinsic fluorescence of 0.1 µM AADC apoenzyme incubated in the presence of PLP at concentrations ranging from 0.005 to 20 μM for 3h at 25°C (in the dark) in 100 mM potassium phosphate buffer pH 7.4.
The data were fitted to the following equation:
where [E]t and [PLP]t represent the total concentrations of the enzyme and PLP, respectively, Y refers to the intrinsic quenching changes at a PLP concentration, and Ymax refers to the fluorescence changes when all enzyme molecules are complexed with coenzyme. Curves fitting was performed using Origin® 9.1 Pro (OriginLab).
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