Expression and purification of Xenopus laevis core histone H2A, H2B, H3, and H4 and subsequent assembly of a histone octamer were performed following published protocols.39 (link) H1G101C, a mutant of X. laevis linker histone H1° (termed H1 in this paper), was expressed and purified according to a previously published method.40 After purification with a cation exchange column (Bio-Rex 70, 50–100 mesh, Bio-Rad Laboratories Inc., product no. 142-5832), H1G101C was labeled with the Cy5 maleimide monoreactive dye (GE Healthcare, product no. PA25031) according to the instructions recommended by the manufacturer. The labeling reaction mixture was purified with another cation exchange column (Bio-Rex 70, 100–200 mesh, Bio-Rad Laboratories Inc., product no. 142-5842) to remove any free dye. The labeling efficiency was close to 100% according to the absorbance values at 280 and 650 nm utilizing a correction factor of 0.05 for the dye at 280 nm. N-Terminally His6-tagged yeast Nap1 (termed Nap1 in this paper) was overexpressed and purified according to previously published protocols.41 (link) Briefly, yNap1 was purified with a Ni-NTA column (HisPur Ni-NTA, Thermo Scientific, product no. 88222) and a Mono-Q anion exchange column (Mono Q 4.6/100 PE, GE Healthcare, product code 17-5179-01).
Cy5 maleimide monoreactive dye
Manufactured by GE Healthcare
Cy5 maleimide monoreactive dye is a fluorescent labeling agent used in various life science applications. It is a cyanine-based dye that can be covalently attached to sulfhydryl groups, enabling the labeling of biomolecules such as proteins, peptides, and antibodies. The dye has a maximum excitation wavelength of approximately 650 nm and a maximum emission wavelength of around 670 nm, making it suitable for detection and analysis using fluorescence-based techniques.
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Lab products found in correlation
Bio rex 70, by Bio-Rad (1 mentions)
Hispur ni nta, by Thermo Fisher Scientific (1 mentions)
Monoq 4.6 100 pe, by GE Healthcare (1 mentions)
Alexa 488 c5 maleimide, by Thermo Fisher Scientific (1 mentions)
C4 column, by Grace Bio-Labs (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Cy3 maleimide, by GE Healthcare (1 mentions)
G25 minitrap column, by GE Healthcare (1 mentions)
Alexa fluor 488 c5 maleimide, by Thermo Fisher Scientific (1 mentions)
4 protocols using cy5 maleimide monoreactive dye
1
Purification and Labeling of Histones
2
Fluorescent Labeling of Histone Proteins
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To generate Alexa Fluor 488 labeled histone H2B (H2B-Alexa488), histone H2B with T112C mutation was dissolved in reaction buffer (25 mM HEPES, pH 7.5, 6 M guanidine, 5 mM TCEP) to a final concentration of 0.5 mM. Then five molar equivalents of Alexa-488-C5-maleimide (Invitrogen) was added to the reaction mixture. Keep the reaction for 4 h at 37°C. The fluorophore labeled histone was purified by HPLC using preparative C4 column (25 mm × 250 mm, 10 μm, Grace). The purity and identity of the product was confirmed by LC–MS. Deconvolution result was obtained by UniDec software (25 (link)) (Supplementary Figure S4 ). Same method was applied to generate Cy5 (Cy5 Maleimide Mono-reactive Dye, GE) labeled histone H2A at K119 position (Supplementary Figure S5 ).
3
Labeling RpoD Protein with Cy5
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E. coli RpoD was labeled with Cy5 at the 366th residue Cys substituting Ser, while Cys residues at the 132nd, 291st, and 295th positions had been substituted for Ser.16 (link) A single-Cys derivative of RpoD was expressed with an N-terminal His6 tag from pRPODS366C, a gift of Prof. Robert Landick at the University of Wisconsin, USA, in E. coli BL21(DE3) at 25 °C and purified as described above for unmodified RpoD. The single-Cys RpoD was incubated with Cy5 maleimide mono-reactive dye (1 mM, GE Healthcare) in the storage buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.1 mM EDTA) overnight at room temperature. Unreacted Cy5-maleimide molecules were removed using an Amicon ultra centrifugal filter (Merck).
4
Fluorescent Labeling of BLS Proteins
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BLSDR-C and BLSKE-C were labeled with (i) Alexa Fluor 488C5 Maleimide and Alexa Fluor 555C5 Maleimide (Thermo Fisher Scientific), and (ii) Cy3 Maleimide and Cy5 Maleimide Mono-Reactive Dye (GE Healthcare), following the manufacturers' instructions. Briefly, proteins at 100 μM concentration in a Tris–HCl 50 mM buffer pH 7.3, supplemented with 1 mM of dithiothreitol (DTT) or 0.7 mM Tris(2-carboxyethyl)phosphine (TCEP), were incubated overnight at 4 °C in the presence of 2 M of the maleimide fluorophores. The reactions were stopped with the addition of β-mercaptoethanol. Then, the unbound dyes were removed using a G25 minitrap column (GE Healthcare). Finally, the labeled proteins were concentrated to ∼50 mg ml−1 stocks and stored at −80 °C for further use. The resulting labeled proteins were named A488BLSDR, A555BLSKE, Cy3BLSDR and Cy5BLSKE, respectively.
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