Alexa Fluor 488 C5 maleimide (Thermo Fisher Scientific), iodoacetyl-N′-7-nitrobenz-2-oxa-1,3-diazol-4-yl (IANBD) amide (Thermo Fisher Scientific), and BADAN (Thermo Fisher Scientific) were used for specific labeling of the different single-cysteine mutants. Proteins were incubated with a fivefold molar excess of the dye overnight at 4 °C. Unreacted dye was removed from the labeled protein with a PD-10 desalting column (GE Healthcare) using 20 mM Hepes, 150 mM KCl, and 0.1 mM TCEP (pH 7.4).
Badan
Manufactured by Thermo Fisher Scientific
BADAN is a fluorescent probe used for the measurement of protein conformational changes. It functions by undergoing changes in fluorescent properties upon binding to proteins, allowing for the monitoring of protein structural dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Pd 10 desalting column, by GE Healthcare (3 mentions)
Alexa fluor 488 c5 maleimide, by Thermo Fisher Scientific (2 mentions)
Oregon green 488 maleimide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 c5 maleimide, by Thermo Fisher Scientific (1 mentions)
Texas red c2 maleimide, by Thermo Fisher Scientific (1 mentions)
Ianbd amide, by Thermo Fisher Scientific (1 mentions)
Fluorolog 3 spectrophotometer, by Horiba (1 mentions)
Ianbd, by Thermo Fisher Scientific (1 mentions)
5 protocols using badan
1
Fluorescent Protein Labeling Protocol
2
Cysteine Labeling of Proteins
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Texas Red C2 Maleimide (Thermo Fisher Scientific), Oregon Green® 488 Maleimide (Thermo Fisher Scientific), Alexa Fluor 488 C5 Maleimide (Thermo Fisher Scientific), Alexa Fluor 546 C5 Maleimide (Thermo Fisher Scientific) and BADAN (Thermo Fisher Scientific) were used for specific labelling of the cysteine mutants. Proteins were incubated with a fivefold molar excess of the dye while gently shaking overnight at 4 °C. Unreacted dye was removed from the labeled protein with a PD-10 desalting column (GE Healthcare) using HP150 buffer with 0.1 mM TCEP.
3
Cysteine Labeling and Lipid Binding Assay
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IANBD amide and BADAN (Thermo Fisher Scientific) were used for specific labeling of the cysteine mutants. Proteins were incubated with a fivefold molar excess of the dye while gently shaking overnight at 4 °C. Unreacted dye was removed from the labeled protein with a PD-10 desalting column (GE Healthcare) using 20 mM Hepes, 150 mM KCl, and 0.1 mM TCEP (pH 7.4). To induce binding, 0.5 μM labeled protein was mixed with ∼500 μM total lipid in 20 mM Hepes, 150 mM KCl, and 0.1 mM TCEP (pH 7.4). The fluorescence emission was measured at 8 °C with a Fluorolog-3 spectrophotometer (model FL322; HORIBA Jobin Yvon) using a 5-nm slit width. The excitation wavelength for IANBD was set at 478 nm, and fluorescence emission was monitored from 500 to 640 nm. For BADAN, the excitation wavelength was set at 380 nm, and fluorescence emission was monitored from 420 to 600 nm. For endoproteinase Glu-C digestion (Sigma–Aldrich), 15 enzyme units were added and incubated for 1 h at room temperature.
4
Fluorescent Labeling of Purified Proteins
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Purified proteins were labelled with environmentally sensitive fluorophores (Table 2 ), either 6-Bromoacetyl-2-Dimethylaminonaphthalene (BADAN) (Thermo Fisher) or N,N’-Dimethyl-N-(iodoacetyl)-N’-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) (Life Technologies) by combining 100 μM protein, 50 μM tris(2-carboxyethyl)phosphine (TCEP), 500 μM fluorophore and PBS to a volume of 500 μl and incubating on ice overnight at 4°C. The protein was then dialysed in PBS using 12kDa dialysis pods (Spectrum Labs) overnight at 4°C. Proteins labelled with Nile blue or Chromis 678 utilised click chemistry. GBP-alkyne was obtained by incubating 100 μM of GBP with 10-fold excess of iodoacetamide alkyne in PBS, 2.5 mM TCEP, pH 7.4, for 2 h at room temperature. Excess of alkyne was removed by extensive dialysis in PBS pH 7.4 at 4°C (Slide-a-Lyser 10 kDa, Pierce). This was then incubated with the dye-azide using the Click-iT® Protein Reaction Buffer kit for 1 h at room temperature. The excess of dye was removed by extensive dialysis as above. Dye protein conjugate concentrations are reported as protein concentration.
5
Protein Expression and Labeling
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