In FCS experiments, the hTRPA1 and the Δ1-688 hTRPA1 N-terminal-His-tag was labeled with the Ni-NTA (Nα,Nα-bis(carboxymethyl)-L-lysine, Nickel(II) Atto647N (Sigma-Aldrich) following the protocol provided by the manufacturer. In FRET experiments, besides the lysine labeling with acceptor Atto647N (Atto-Tech), the N-terminal-His-tag was labeled with the Ni-NTA Atto550 fluorophore (Sigma-Aldrich) following the protocol provided by the manufacturer. In all cases, excess fluorophore was added to the aqueous protein solution containing 50 mM PBS buffer at pH 7.8 supplemented with 130 mM NaCl and 0.014% phosphatidyl choline (FC14) (Sigma) and left for 8 h at 4°C. The labeled protein was subsequently purified using size exclusion chromatography on a Sephadex ™ G-25 Medium column (GE Healthcare, UK). To remove excess free dyes from the labeled proteins and also avoid hydrolysis of extra fluorophore, the samples were dialysed (3k membrane) for two times, four hours of incubation in each time at 4°C. For long storage, the conjugates were divided into small aliquots and frozen at 20°C to avoid repeated freezing and thawing.
Sephadex g 25 medium column
Manufactured by GE Healthcare
Sourced in United Kingdom
Sephadex G-25 medium columns are size-exclusion chromatography columns used for the separation and purification of molecules based on their size and molecular weight. These columns are designed to effectively separate small molecules, such as salts, from larger molecules, like proteins, peptides, or nucleic acids. The Sephadex G-25 medium provides a porous matrix that allows smaller molecules to permeate while retaining larger molecules, enabling efficient separation and purification.
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5 protocols using sephadex g 25 medium column
1
Fluorescent Labeling of TRPA1 Proteins
2
Fluorescent Labeling of Proteins for Biophysical Studies
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In Fluorescence correlation spectroscopy (FCS) experiments, lysines of NpDps and cysteines of NpFdx proteins were labeled with the fluorophore Atto550 (λex 544 nm; λem 576 nm) by following the protocol provided by the manufacturer (Atto-Tec) respectively. For DNA binding experiments, linear DNA was used in all experiments. In Fluorescence resonance energy transfer (FRET) experiments, lysines were labeled with donor Atto550 and cysteines were labeled with acceptor Atto647N (λex 644 nm; λem 661 nm) in NpDps and NpFdx proteins, respectively. In all cases, excess fluorophore was added to the aqueous protein solution and left for 8 h at 4 ºC. The labeled protein was subsequently purified using size exclusion chromatography on a Sephadex ™ G-25
Medium column (GE Healthcare, UK). To remove excess free dyes from the labeled proteins and also avoid hydrolysis of an extra fluorophore, the samples were dialyzed (3k membrane) two times, In all cases, we added 0.1% Tween-20 (Sigma) to the samples in order to diminish surface interactions with the glass coverslip.
Medium column (GE Healthcare, UK). To remove excess free dyes from the labeled proteins and also avoid hydrolysis of an extra fluorophore, the samples were dialyzed (3k membrane) two times, In all cases, we added 0.1% Tween-20 (Sigma) to the samples in order to diminish surface interactions with the glass coverslip.
3
Salt Stress Kinase Activity Assay
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One gram of each fresh plant sample harvested at two and 24 h after the salt treatment, was ground in 1 mL of cold extraction buffer containing 100 mM Tricine-NaOH (pH 8.0), 25 mM NaF, 5 mM dithiothreitol, 2 mM tetrasodium pyrophosphate, 0.5 mM ethylene diamine tetra acetic acid, 0.5 mM ethylene glycol tetra acetic acid, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM protease inhibitor cocktail (Sigma P9599), phosphatase inhibitors (PhosStop; Roche), and insoluble polyvinylpyrrolidone to the solution with a final concentration of 2% (w/v). The homogenate was transferred to two cold microfuge tubes and centrifuged at 12,000×g for 5 min at 4 °C. The supernatant (750 uL) was desalted on a 2.5 mL centrifuge column (Sephadex G-25 medium columns; GE Healthcare) that was pre-equilibrated. SnRKl activity was determined by the Universal Kinase Activity Kit (R&D Systems, Minneapolis, MN, United States, EA004) by using AMARA polypeptide as the substrate [59 (link)].
4
Fruit Tissue Protein Extraction and Kinase Assay
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Fruit tissue (1 g) was ground in 1 ml of cold extraction buffer: 100 mmol l−1 HEPES, pH 8, 25 mmol l−1 NaF, 2 mmol l−1 sodium pyrophosphate, 0.5 mmol l−1 ethylene diamine tetra acetic acid, 0.5 mmol l−1 ethylene glycol tetra acetic acid, 1 mmol l−1 anisole, 5 mmol l−1 dithiothreitol, 25 mmol l−1 β-mercaptoethanol and 1 mol·l−1 pepstatin A. The suspension was transferred to two cold microfuge tubes and clarified by centrifugation for 5 min at 12000 × g at 4 °C. The supernatant (750 μl) was desalted on a 2.5-ml centrifuge column (Sephadex G-25 medium columns; GE Healthcare, Chalfont St Giles, UK) treated with equilibration solution. Using AMARA polypeptide as the substrate (Zhang et al. 2009 (link), Debast et al. 2011 (link)), the SnRKl activity was measured using a Universal Kinase Activity Kit (R & D Systems, Minneapolis, MN, USA).
5
Extraction and Assay of SnRKl Kinase Activity
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Fruit tissue (1 g) was ground in 1 mL of cold extraction buffer consisting of 100 mmol⋅l-1 HEPES, pH 8, 25 mmol⋅l-1 NaF, 2 mmol⋅l-1 sodium pyrophosphate, 0.5 mmol⋅l-1ethylene diamine tetra acetic acid, 0.5 mmol⋅l-1 ethylene glycol tetra acetic acid, 1 mmol⋅l-1 anisole, 5 mmol⋅l-1 dithiothreitol, 25 mmol⋅l-1 β-mercaptoethanol, and 1 mol⋅l-1 pepstatin A. The suspension was transferred to two cold microfuge tubes and clarified by centrifugation for 5 min at 12,000 ×g at 4°C. The supernatant (750 μL) was desalted on a 2.5 mL centrifuge column (Sephadex G-25 medium columns; GE Healthcare, United Kingdom) treated with equilibration solution. Using AMARA polypeptide as the substrate (Zhang et al., 2009 (link)), the SnRKl activity was measured using a Universal Kinase Activity Kit (R&D Systems, Minneapolis, MN, United States).
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