The MFS peptide biotin-N-GGC GS(GPGGA)5GGK YGGS-K(ε-N3) (residues for fluorophore attachment are underlined), MFSC peptide biotin-N-GGGGS(GPGGA)5GGK YC GS-K(ε-N3) and the MFS0 peptide biotin-N-GGGGS (GPGGA)5 GGKYG GS-K(ε-N3) were purified via preparative C18 reverse phase HPLC (5 µm particle size, 250 × 21.2 mm, Agilent) applying a linear gradient of 0.1% triflouracetic acid (TFA) in deionized water (buffer A) and 0.1% TFA, 9.9% deionized water, 90% acetonitrile (buffer B) over 80 min at a flow rate of 5 ml/min. The identity of the spring modules was confirmed via MALDI-TOF mass spectrometry (Bruker). 350 µg lyophilized peptide was dissolved in PBS at a final concentration of 5 mg/ml, mixed with Alexa Fluor 555 maleimide (Thermo Fisher Scientific) at a 1.5-fold molar surplus and then incubated for 2 h followed by an incubation with 1.5-fold excess Alexa Fluor 647 succinimidyl ester (Thermo Fisher Scientific) in the presence of 330 mM NaHCO3 for 2 h at room temperature. HPLC purification and lyophilization was performed after each step and successful conjugation was confirmed via mass spectrometry.
Alexa fluor 555 maleimide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555 maleimide is a fluorescent dye used for labeling and detection in biological applications. It has an excitation maximum at 555 nm and an emission maximum at 565 nm. The maleimide functional group enables covalent labeling of thiol-containing biomolecules such as proteins.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647 maleimide, by Thermo Fisher Scientific (3 mentions)
Sephacryl s 100 resin, by GE Healthcare (2 mentions)
Alexa fluor 647 succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Maldi tof mass spectrometry, by Bruker (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Alexa fluor 488 maleimide, by Thermo Fisher Scientific (1 mentions)
30 kda spin filters, by Merck Group (1 mentions)
E coli reca, by New England Biolabs (1 mentions)
8 protocols using alexa fluor 555 maleimide
1
Site-Specific Labeling of Multifunctional Peptides
2
Fluorescent Labeling of E. coli Topoisomerase I
3
Fluorescent Labeling of Ykt6 Proteins
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The 25 μM double cysteine mutants of yYkt6 or rYkt6 proteins were incubated with 100 μM donor (Alexa Fluor 555 Maleimide, Thermo Fisher Scientific Inc), and 200 μM acceptor (Alexa Fluor 647 Maleimide, Thermo Fisher Scientific Inc) in the presence of 1 mM Tris (2-carboxyethyl) phosphine hydrochloride at 4 °C overnight. Afterward, the unreacted dyes were removed from the protein solution by SEC using a normal buffer containing 1 mM β-mercaptoethanol.
4
Fluorescent Labeling of E. coli Topoisomerase I
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E. coli Topoisomerase I (EcTopoI) was purified as previously described28 (link) and stored at −20°C at 2 mg/mL in storage buffer (50 mM Tris-HCl pH8, 1mM DTT, 50 mM KCl, 50% glycerol). To create fluorescently labeled EcTopoI, the purified protein was dialyzed into de-gassed labeling buffer (50 mM Tris-HCl pH 7, 150 mM KCl, 1 mM EDTA). Protein was then incubated overnight at 4°C with 10-fold molar excess of Alexa Fluor 555 maleimide (A20346, Thermo Fisher, Waltham, MA). After incubation, protein and dye were purified over a Sephacryl S-100 resin (17-1194-01, GE Healthcare Life Sciences) to remove excess dye. After purification mass spectrometry confirmed primarily single labeling of the protein, although the location of the label is unknown. Bulk activity assays confirmed relaxation activity for Alexa 555 labeled EcTopoI, although at approximately five times lower rate than wild type EcTopoI. For the experiments, EcTopoI was first diluted 20-fold in buffer (50 mM Tris-HCl pH8, 150 mM KCl, 1mM EDTA) and stored on ice until needed. Immediately prior to flowing protein into the flow cell, EcTopoI was further diluted to 40 nM in the single molecule fluorescence oxygen scavenging buffer, described below. For experiments with Alexa 555 labeled EcTopoI, the protein was diluted to 2 nM.
5
Redox Immunohistochemistry for Thiol Labeling
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Assays were performed as previously described, according to a procedure we refer to as redox immunohistochemistry [26 (link),27 (link)]. Briefly, equal amounts of cells were harvested and suspended in a lysis solution containing Tris-HCl 50 mM, SDS 1%, EDTA 1 mM, NEM 10 mM (N-Ethylmaleimide, Sigma-Aldrich, St. Louis, Mo, USA, E3876), Alexa Fluor 647 Maleimide 20 uM (Thermo Scientific™, Waltham, MA, USA, A20347), and proteinase inhibitors mix to label reduced thiols. Lysates were incubated for 5 min at 70 °C, sonicated, and finally incubated for 30 min at RT in the dark. Quenching of unreacted thiols was performed with 100 mM NEM for 30 min. Protein extracts were then precipitated with a cold precipitation solution (50% acetone, 25% methanol, and 25% ethanol). Next, disulfide bonds were reduced with 20 mM TCEP (Tris(2-carboxyethyl)phosphine, Thermo Scientific™, Waltham, MA, USA, 20490). Newly reduced thiols were labelled with AlexaFluor 555 Maleimide 20 uM (Thermo Scientific™, Waltham, MA, USA, A20346). After precipitation, samples were diluted in Laemmli sample buffer, and proteins were resolved under reducing conditions. After electrophoresis, the gel was fixed with 5orthophosphoric 2.5% orthophosphoric acid overnight under mild agitation. Final images and analysis were performed with the Chemidoc MP Imaging System (Bio-Rad Laboratories S.r.l., Hercules, CA, USA).
6
Fluorescent Labeling and Encapsulation of Nanoparticles
7
Purification and Fluorescent Labeling of Clathrin and Adaptor Protein Complexes
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Clathrin and AP fractions were purified as previously described (Campbell et al., 1984 (link); Ahle and Ungewickell, 1986 (link); Wu et al., 2010 (link)) from the coat fraction by gel filtration through a Tricon 10/300 Superose 6 column (GE Healthcare Life Sciences). Fractions containing clathrin or AP fraction (primarily consisting of AP2, but also containing small percentages of AP180 [Ahle and Ungewickell, 1986 (link)], AP1 [Keen, 1987 (link)], and Auxilin [Ahle and Ungewickell, 1990 (link)]) were pooled separately, dialyzed, and concentrated before being flash frozen in liquid nitrogen and stored at −80°C.
Purified clathrin was conjugated with Alexa fluorophores as described previously (Wu et al., 2010 (link)). Alexa Fluor 555 maleimide (Invitrogen A20346) and Alexa Fluor 647 maleimide (Invitrogen A20347) were used to label the purified clathrin. Conjugated clathrin were subjected to a round of assembly and disassembly to ensure functionality. The protein and dye concentrations were estimated from the absorbance at 280 nm, 555 nm, or 647 nm, respectively. The ratio of dye to protein was then calculated for each batch; values were typically in the range of 2.5–3.5 dyes per protein molecule.
Purified clathrin was conjugated with Alexa fluorophores as described previously (Wu et al., 2010 (link)). Alexa Fluor 555 maleimide (Invitrogen A20346) and Alexa Fluor 647 maleimide (Invitrogen A20347) were used to label the purified clathrin. Conjugated clathrin were subjected to a round of assembly and disassembly to ensure functionality. The protein and dye concentrations were estimated from the absorbance at 280 nm, 555 nm, or 647 nm, respectively. The ratio of dye to protein was then calculated for each batch; values were typically in the range of 2.5–3.5 dyes per protein molecule.
8
Purification of Fluorescent E. coli SSB
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Both wild-type and fluorescently labeled E. coli SSB were expressed and purified as described previously (Lohman et al., 1986 (link); Roy et al., 2009 (link)), with an addition of a dsDNA cellulose column to remove a minor exonuclease contaminant (Bujalowski and Lohman, 1991b (link)). The labeled SSB was single-point mutated from Ala to Cys at position 122 in the C-terminus, and labeled with AlexaFluor555 maleimide (Invitrogen, Grand Island, NY) to the extent of ∼25% (∼1 dye per tetramer) as described previously (Roy et al., 2009 (link)). E. coli RecA was purchased from New England Biolabs (M0249S; Ipswich, MA).
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