HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium bicarbonate (NaHCO3) agarose, streptavidin agarose beads, isopropyl β-d -1-thiogalactopyranoside (IPTG) and ampicillin were obtained from Sigma-Aldrich. The polymer wrapping the QD was synthesized according to our previous study.28 (link) Oligonucleotides were bought from IDT Technologies. SulfoCy5 maleimide was bought from Lumiprobe, while Texas Red maleimide was bought from Vector Laboratories. HEPES 1× is a solution of 25 mM of HEPES and 150 mM of NaCl, adjusted to pH 7.6.
Sulfo cy5 maleimide
Manufactured by Lumiprobe
Sourced in Germany
Sulfo-Cy5 maleimide is a fluorescent labeling reagent that can be used to conjugate biomolecules such as proteins, peptides, and other thiol-containing compounds. It contains a sulfo-NHS ester group that reacts with primary amines and a maleimide group that reacts with sulfhydryl groups.
Automatically generated - may contain errors
Lab products found in correlation
Sulfo cy3 maleimide, by Lumiprobe (3 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Streptavidin agarose beads, by Merck Group (1 mentions)
Texas red maleimide, by Vector Laboratories (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fitc maleimide, by Lumiprobe (1 mentions)
6 protocols using sulfo cy5 maleimide
1
Quantum Dot Labeling Protocol
2
Fluorescent Labeling of Lactaptin Analogs
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The recombinant lactaptin analogs RL2, Т1_RL and Т2_RL were isolated from E. coli and purified according to a protocol for the isolation of RL2 (5 (link)).
To label RL2, T1_RL and T2_RL with sulfo-Cy5, 0.2 mM of each protein was incubated with 1.25 mM of the fluorescent dye sulfo-Cy5 maleimide (Ex/Em=646/662; Lumiprobe GmbH, Hannover, Germany) in Tris-HCl buffer (pH 5.5) for 4 h at 25°С. The purification of the conjugates RL2-Cy5, T1_RL-Cy5 and T2_RL-Cy5 was performed by reversed-phase high-performance liquid chromatography on a С18 column using a Milichrom A-02 chromatograph (EcoNova, Novosibirsk, Russia) with acetonitrile-water as a mobile phase.
To label RL2, T1_RL and T2_RL with sulfo-Cy5, 0.2 mM of each protein was incubated with 1.25 mM of the fluorescent dye sulfo-Cy5 maleimide (Ex/Em=646/662; Lumiprobe GmbH, Hannover, Germany) in Tris-HCl buffer (pH 5.5) for 4 h at 25°С. The purification of the conjugates RL2-Cy5, T1_RL-Cy5 and T2_RL-Cy5 was performed by reversed-phase high-performance liquid chromatography on a С18 column using a Milichrom A-02 chromatograph (EcoNova, Novosibirsk, Russia) with acetonitrile-water as a mobile phase.
3
Protein Labeling with Fluorescent Dyes
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To label the proteins with dye, a 1:1 mixture of sulfo-Cy3 maleimide (Lumiprobe) and sulfo-Cy5 maleimide (Lumiprobe) was added to the proteins at a protein:fluorophore molar ratio of 1:10. The labeling reaction was performed at 4 °C for 2 h. The labeled proteins were then diluted and added to anti-FLAG resin, and the excess free dyes were effectively removed by extensive washing29 (link). The proteins were eluted by elution buffer and concentrated to 0.3–0.4 mg ml−1 (Supplementary Fig. S2 b).
4
Site-specific labeling of ubiquitin and Ufd1
5
Fluorescent Labeling of PRE-h Proteins
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Lyophilized
PRE-h-36, PRE-h-58, and PRE-h-41 were dissolved at 1.5 mg/mL in PBS140 pH
7.0 in 500 μL. 10-fold molar excess freshly prepared TCEP (Sigma-Aldrich)
was added and incubated for >15 min at room temperature. Next,
10-fold
molar excess sulfo-Cy5 maleimide (Lumiprobe GmbH) sulfo-Cy3 maleimide
(Lumiprobe GmbH) and FITC maleimide (Lumiprobe GmbH) from a 10 mg/mL
stock in DMSO (Sigma-Aldrich) was added dropwise to the proteins to
yield Cy5-PRE-h-36, Cy3-PRE-h-58,
and FITC-PRE-h-41. The reaction mixture was wrapped
in aluminum foil and incubated in a rocker for 2 h at room temperature
followed by overnight incubation at 4 °C. Excess dye was quenched
by addition of 1 mM DTT and extensively removed by multiple rounds
of dilution of the labeled conjugate with PBS140 followed
by 10 kDa spin concentrations (Amicon), until no absorbance could
be measured by UV–vis in the eluate.
PRE-h-36, PRE-h-58, and PRE-h-41 were dissolved at 1.5 mg/mL in PBS140 pH
7.0 in 500 μL. 10-fold molar excess freshly prepared TCEP (Sigma-Aldrich)
was added and incubated for >15 min at room temperature. Next,
10-fold
molar excess sulfo-Cy5 maleimide (Lumiprobe GmbH) sulfo-Cy3 maleimide
(Lumiprobe GmbH) and FITC maleimide (Lumiprobe GmbH) from a 10 mg/mL
stock in DMSO (Sigma-Aldrich) was added dropwise to the proteins to
yield Cy5-PRE-h-36, Cy3-PRE-h-58,
and FITC-PRE-h-41. The reaction mixture was wrapped
in aluminum foil and incubated in a rocker for 2 h at room temperature
followed by overnight incubation at 4 °C. Excess dye was quenched
by addition of 1 mM DTT and extensively removed by multiple rounds
of dilution of the labeled conjugate with PBS140 followed
by 10 kDa spin concentrations (Amicon), until no absorbance could
be measured by UV–vis in the eluate.
6
Cysteine Labeling of TmrAB Protein
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The single natural cysteine in wild-type TmrAB at position 416 in TmrA was labeled by
maleimide chemistry. Therefore, TmrAB was incubated with sulfoCy5 maleimide (Lumiprobe) in
PBS pH 6.8 for 1 h at room temperature. A protein concentration of 1 mg/mL and 20-fold
molar excess of sulfoCy5 maleimide were used. Subsequently, the reaction was quenched with
1 mM β-mercaptoethanol. Dye excess was removed via size-exclusion
spin columns (Bio-Rad) followed by size-exclusion chromatography (SEC).
maleimide chemistry. Therefore, TmrAB was incubated with sulfoCy5 maleimide (Lumiprobe) in
PBS pH 6.8 for 1 h at room temperature. A protein concentration of 1 mg/mL and 20-fold
molar excess of sulfoCy5 maleimide were used. Subsequently, the reaction was quenched with
1 mM β-mercaptoethanol. Dye excess was removed via size-exclusion
spin columns (Bio-Rad) followed by size-exclusion chromatography (SEC).
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