Labeling of a Protein with Fluorophores Using Maleimide Derivitization

Nanda, Jagpreet S.; Lorsch, Jon R.

doi:10.1016/b978-0-12-420070-8.00007-6

Cited by 34 publications

(30 citation statements)

References 1 publication

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“…It seems clear that this methodology could be applied with a high probability of success to conveniently conjugate meso-Cl near-IR dyes to antibodies, monobodies, and nanobodies to form selective agents for optical imaging in vivo. Traditional conjugation techniques tend to require modification of the dye to include maleimide or succinimide functionality [25,31,32], but the method developed here circumvents that process.…”

Section: Discussionmentioning

confidence: 99%

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Site-Specific Labeling of Proteins with Near-IR Dyes

Lin¹,

Usama²,

Burgess³

2018

Preprint

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Convenient labeling of proteins is important for observing its function under physiological conditions.  In tissues particularly, heptamethine cyanine dyes (Cy-7) are valuable because they absorb in near infrared (NIR) region (750 – 900 nm) where light penetration is maximal.  In this work, we found Cy-7 dyes with a meso-Cl functionality covalently binding to proteins with free Cys residues under physiological conditions (aqueous environments, at near neutral pH, and 37 °C).  It transpired that the meso-Cl of the dye was displaced by free thiols in protein, while nucleophilic side-chains from amino acids like Tyr, Lys, and Ser did not react.  This finding shows a new possibility for convenient and selective labeling of proteins with near-IR fluorescent probes.

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Section: Discussionmentioning

confidence: 99%

“…Competition study of MHI-148 with amino acids in aqueous buffer Blocking experiments were performed to be absolutely sure that the vimentin Cys was the reactive group for coupling to MHI-148 in aqueous buffer at 37 °C. Thus, maleimide-blocked protein[24,25] was formed by incubating vimentin with 6-maleimidohexanoic acid (6-MA; 18 h in…”

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confidence: 99%

Site-Specific Labeling of Proteins with Near-IR Dyes

Lin¹,

Usama²,

Burgess³

2018

Preprint

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“…The eIF5 mutants were expressed in BL21(DE3) Codon Plus cells (Stratagene) as described (32). The purified proteins were then fluorescently labeled with fluorescein-maleimide (34).…”

Section: Methodsmentioning

confidence: 99%

“…Next we generated various single cysteine mutants of Cys-lite eIF5 by introducing cysteines at non-conserved, surface-exposed positions in the NTD, CTD, and linker region using sitedirected mutagenesis (Table 1). These mutants were then expressed in Escherichia coli and purified, and the cysteine residues were labeled with fluorescein-maleimide (34). We also labeled the three naturally occurring cysteines at positions 6, 289, and 294 in variants containing each one as the only surfaceexposed cysteine (Table 1).…”

Section: The Ctt Of Eif1a and N-terminal Domain Of Eif5 Move Closer Tmentioning

confidence: 99%

Coordinated Movements of Eukaryotic Translation Initiation Factors eIF1, eIF1A, and eIF5 Trigger Phosphate Release from eIF2 in Response to Start Codon Recognition by the Ribosomal Preinitiation Complex*

Nanda

Saini

Munoz

et al. 2013

Journal of Biological Chemistry

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124

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Background: Start codon recognition triggers eIF1 and P i release from the preinitiation complex. Results: The C-terminal tail of eIF1A moves closer to eIF5 upon start codon recognition, and this movement is required for P i release. Conclusion: eIF1 release and movement of the eIF1A C-terminal tail toward eIF5 are coupled processes. Significance: Start codon recognition induces coordinated movements of initiation factors that trigger downstream events.

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“…In this approach, a free thiol group (i.e., cysteine) on the protein is reacted with a fluorophore that is derivatized with a maleimide group, forming a thioether bond. It is a selective reaction around pH 7.0, though above pH 8.0, primary amines are also labeled (Nanda & Lorsch, 2014).…”

Section: Strategic Planningmentioning

confidence: 99%

Ratiometric Single‐Molecule FRET Measurements to Probe Conformational Subpopulations of Intrinsically Disordered Proteins

Nasir

Bentley

Deniz

2020

CP Chemical Biology

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Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid‐liquid phase separation. The so‐called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein “structure‐function paradigm,” as their functions do not necessarily involve well‐defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single‐molecule fluorescence methods can overcome this ensemble‐average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single‐molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in‐depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel

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Labeling of a Protein with Fluorophores Using Maleimide Derivitization

Cited by 34 publications

References 1 publication

Site-Specific Labeling of Proteins with Near-IR Dyes

Site-Specific Labeling of Proteins with Near-IR Dyes

Coordinated Movements of Eukaryotic Translation Initiation Factors eIF1, eIF1A, and eIF5 Trigger Phosphate Release from eIF2 in Response to Start Codon Recognition by the Ribosomal Preinitiation Complex*

Ratiometric Single‐Molecule FRET Measurements to Probe Conformational Subpopulations of Intrinsically Disordered Proteins

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