Atto532 maleimide

Manufactured by ATTO-TEC

Sourced in Germany

The Atto532 maleimide is a fluorescent dye used for labeling biomolecules. It has an excitation maximum at 532 nm and an emission maximum at 553 nm, making it suitable for use with common laser sources. The maleimide functional group allows for covalent attachment to thiol-containing molecules, such as proteins and peptides.

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Lab products found in correlation

Nanodrop 2000, by Thermo Fisher Scientific (4 mentions) Pd minitrap g 25 prepacked columns, by GE Healthcare (2 mentions) Atto488 maleimide, by ATTO-TEC (2 mentions) Alexa 488 maleimide, by Thermo Fisher Scientific (1 mentions) 1 4 dithiothreitol dtt, by Merck Group (1 mentions)

4 protocols using atto532 maleimide

Fluorescent Labeling of Ubiquitin Sensors

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mPUP (50 μM), pPUPs (50 to 60 μM), or Ub(S20C) (50 μM) in 50 mM Hepes (pH 7.5), 100 mM NaCl, and 1 mM TCEP were incubated with Alexa488-maleimide (75 μM; Thermo Scientific) or Atto532-maleimide (75 to 90 μM; ATTO-TEC GmbH) at room temperature for 2 hours to label C80 of the RUZ domain in mPUP with Alexa488, C39 of pUBD in pPUPs with Atto532, or Ub(S20C) with Atto532. Subsequently, 10 mM 2-mercaptoethanol was added to consume unreacted maleimide dyes, and the proteins were purified by IMAC to remove the unbound dye. The degree of labeling (DOL) and concentrations of the labeled sensors were calculated using the equations below

DOL = \frac{A_{m} \times ε_{prot}}{(A_{280} - A_{m} \times {CF}_{280}) \times ε_{m}} {CF}_{280} = \frac{ε_{280}}{ε_{m}}

Protein concentration (M) = \frac{[A_{280} - (A_{m} \times {CF}_{280})]}{ε_{prot}}

Here, A_m represents the absorbance at the dye absorption maximum, A₂₈₀ is absorbance at 280 nm of the labeled protein, ɛ_prot is the unlabeled protein extinction coefficient at 280 nm, ɛ₂₈₀ is the extinction coefficient at 280 nm of the dye alone, ɛ_m is the extinction coefficient at the absorption maximum of the dye, and CF₂₈₀ is the correction factor at 280 nm. The DOL obtained for the Alexa488-labeled mPUP and Atto532-labeled pPUPs typically were 55 ± 10%.

Yakoub G., Choi Y.S., Wong R.P., Strauch T., Ann K.J., Cohen R.E, & Ulrich H.D. (2023). Avidity-based biosensors for ubiquitylated PCNA reveal choreography of DNA damage bypass. Science Advances, 9(36), eadf3041.

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Fluorescent Labeling of ENTH Proteins

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The ENTH WT (C96A A155C) domain and the ENTH R114A (C96A A155C) were labeled with Atto488 maleimide (or Atto532 maleimide) (ATTO-TEC GmbH, Siegen, Germany) by cysteine modification. The substitution reaction took place in PBS (136 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.8 mM K₂HPO₄, pH 7.4) with a molar access of the fluorophore of 1.4 in respect to the protein. The substitution was performed at 4 °C with gentle agitation on a tumbling shaker for 16 h.
Proteins were separated from free fluorophores by size-exclusion chromatography with a PD MiniTrap G-25 prepacked columns (GE Healthcare Life Science, Chalfont St Giles, UK). Finally, the degree of labeling (DoL) of the proteins was determined spectrophotometrically by measuring the UV–Vis (NanoDrop 2000, Thermo Fisher Scientific Inc., Waltham, USA). For this purpose, the following equation was used:

DoL = A_{max} \times ε_{prot} \times (A_{280} - {(A_{max} \times {CF}_{280})}^{- 1} \times ε_{max}^{- 1} .

A_max is the absorption at the characteristic wavelength of the fluorophore, ε_max is the molar extinction coefficient of the fluorophore, A₂₈₀ is the absorption of the protein at 280 nm, ε_prot is the molar extinction coefficient of the labeled protein and CF₂₈₀ is the correction factor of the fluorophore.

Kroppen B., Teske N., Yambire K.F., Denkert N., Mukherjee I., Tarasenko D., Jaipuria G., Zweckstetter M., Milosevic I., Steinem C, & Meinecke M. (2020). Cooperativity of membrane-protein and protein–protein interactions control membrane remodeling by epsin 1 and affects clathrin-mediated endocytosis. Cellular and Molecular Life Sciences, 78(5), 2355-2370.

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Labeling Biomolecules with ATTO Dyes

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All
chemicals were purchased with the highest
purity available. OM, NM, DM, UM, DDM, and DPC were from Glycon Biochemicals
(Luckenwalde, Germany) or Anatrace (Maumee, USA). LDAO was from Anatrace,
Tris from Carl Roth (Karlsruhe, Germany), NaCl (AnalaR Normapur) from
VWR (Darmstadt, Germany) or Carl Roth, 1,4-dithiothreitol (DTT) from
Sigma–Aldrich (Steinheim, Germany), and N-propargyl-L-lysine
(PrK) from SiChem (Bremen, Germany). ATTO532 maleimide and ATTO647N
azide were from Atto-Tec (Siegen, Germany).

Frotscher E., Krainer G., Hartmann A., Schlierf M, & Keller S. (2018). Conformational Dynamics Govern the Free-Energy Landscape of a Membrane-Interacting Protein. ACS Omega, 3(9), 12026-12032.

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Fluorescent Labeling of ENTH Proteins

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The ENTH WT (C96A A155C) domain and the ENTH R114A (C96A A155C) were labeled with Atto488 maleimide (or Atto532 maleimide) (ATTO-TEC GmbH, Siegen, Germany) by cysteine modification. The substitution reaction took place in PBS (136 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.8 mM K2HPO4, pH 7.4) with a molar access of the fluorophore of 1.4 in respect to the protein. The substitution was performed at 4°C with gentle agitation on a tumbling shaker during 16 h.
Proteins were finally separated from free fluorophores by size exclusion chromatography with a PD MiniTrap G-25 prepacked columns (GE Healthcare Life Science, Chalfont St Giles, UK). Finally, the degree of labeling (DoL) of the proteins was determined spectrophotometrically by measuring the UV-Vis (NanoDrop 2000, Thermo Fisher Scientific Inc., Waltham, USA). For this purpose, the following equation was used:
Amax is the absorption at the characteristic wavelength of the fluorophore, εmax is the molar extinction coefficient of the fluorophore, A280 is the absorption of the protein at 280 nm, εprot is the molar extinction coefficient of the labeled protein and CF280 is the correction factor of the fluorophore.

Kroppen B., Teske N., Yambire K.F., Denkert N., Murkhejee I., Tarasenko D., Jaipuria G., Zweckstetter M., Milošević I., Steinem C., & Meinecke M. (2020). Cooperativity of membrane-protein and protein-protein interactions control membrane remodeling by epsin 1 and regulate clathrin-mediated endocytosis.

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