Cyanine5 Boc-hydrazide, 5 mg

Cyanine5 Boc-hydrazide 25 mg
Cyanine5 Boc-hydrazide 25 mg

Cy5 Boc-hydrazide

Cy5 Boc-hydrazide is a boc-protected reactive dye containing a Boc-protected hydrazide. Boc protective group can be removed with TFA, or other strong acid to form the reactive hydrazide, which can then couple with various carbonyl groups yielding Cyanine5-labeled molecules.

  • Catalog: BP-22573

  • Name: Cy5 Boc-hydrazide

  • Formula: C37H49ClN4O3

  • MW: 633.3

  • CAS:

  • Purity   96%

  • Ships Within: 24 Hours

  • Storage Condition: -20°C

  • Solubility: DMSO, DMF, DCM

  • Shipping: Ambient temperature

 

DESCRIPTION

Application

18:1 Cy5 PE or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5), chloroform has been used:

  • as a standard for the estimation of absolute density of Cy5 labeled Trx-His6-NCav-CT on supported lipid bilayers (SLB)
  • in annexin-coated vesicle production
  • to label vesicle types in order to optically distinguish them from other vesicles and to initiate biochemical reactions in a cell-mimetic compartment

Biochem/physiol Actions

Fluorescent labelled lipids are generally useful in cell biology and biophysical studies. Cellular events involving lipids such as lipid domain generation, disaggregation and re-organization can be studied with fluorescent lipids. They are also useful in analyzing the physical properties of lipid bilayers.

Packaging

5 mL Amber Glass Screw Cap Vial (810335C-5mg)
5 mL Amber Glass Screw Cap Vial (810335C-1mg)

SAFETY INFORMATION

Pictograms

GHS06,GHS08

Signal Word

Danger

Hazard Statements

H302 – H315 – H319 – H331 – H336 – H351 – H361d – H372 – H412

Precautionary Statements

P201 – P273 – P301 + P312 + P330 – P302 + P352 – P304 + P340 + P311 – P308 + P313

Hazard Classifications

Acute Tox. 3 Inhalation – Acute Tox. 4 Oral – Aquatic Chronic 3 – Carc. 2 – Eye Irrit. 2 – Repr. 2 – Skin Irrit. 2 – STOT RE 1 – STOT SE 3

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Target Organs

Central nervous system

Storage Class Code

6.1D – Non-combustible, acute toxic Cat.3 / toxic hazardous materials or hazardous materials causing chronic effects.

Cyanine5 Boc-hydrazide 25 mg
Cyanine5 Boc-hydrazide 25 mg

WGK

WGK 3

Flash Point(F)

does not flash

Flash Point(C)

does not flash

Cy5 dye (also sold under Sulfo-Cyanine5 name) is one of the most popular far-red fluorescent dyes. It is often a reagent of choice for protein and peptide labeling. Cy5 dye is a water-soluble, bright, far-red-fluorescent dye with excitation ideally suited for the 633 nm or 647 nm laser lines. Cy5 conjugates of antibodies, peptides, and proteins are pH insensitive from pH 4 to pH 10. A significant advantage to using long wavelength dyes such as Cy5 or AF 647 dye over other fluorophores is the low autofluorescence of biological specimens in this region of the spectrum.
The NHS ester (or succinimidyl ester) is the most popular amine reactive group for labeling with the primary amines of proteins (Lys), amine-modified oligonucleotides, and other amine-containing molecules. We do not recommend using Cy5 NHS ester for labeling proteins at high molar ration due significant self-quenching. Cy5 conjugates are recommended for detection of moderate-to-high abundance targets. For detection of low-abundance biological targets we recommend using APDye 647 (Alexa Fluor® 647 analog), or IR 650 dye (IRDye 650 analog), which can be attached to proteins at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection.

AxisPharm offers 3000+ PEG Linkers with high purity. Different kinds of PEG Reagents may be available by custom synthesis.

Abstract
In-solution conjugation is the most commonly used strategy to label peptides and proteins with fluorophores. However, lack of site-specific control and high costs of fluorophores are recognised limitations of this approach. Here, we established facile access to grams of Cy5-COOH via a two-step synthetic route, demonstrated that Cy5 is stable to HF treatment and therefore compatible with tert-butyloxycarbonyl solid phase peptide synthesis (Boc-SPPS), and coupled Cy5 to the N-terminus of α-conotoxin RgIA while still attached to the resin. Folding of the two-disulfide containing Cy5-RgIA benefitted from the hydrophobic nature of Cy5, resulting in only the globular disulfide bond isomer. In contrast, wild-type α-RgIA folded into the inactive ribbon and bioactive globular isomer under the same conditions. Labelled α-RgIA retained its ability to inhibit acetylcholine (100 µM)-evoked current reversibly with an IC of 5.0 nM (Hill coefficient = 1.7) for Cy5-RgIA and an IC of 1.6 (Hill coefficient = 1.2) for α-RgIA at the α9α10 nicotinic acetylcholine receptor (nAChR) heterologously expressed in Xenopus oocytes.
Cy5-RgIA was then used to successfully visualise nAChRs in the RAW264.7 mouse macrophage cell line. This work introduced not only a new and valuable nAChR probe, but also a new versatile synthetic strategy that facilitates production of milligram to gram quantities of fluorophore-labelled peptides at low cost, which is often required for in vivo experiments. The strategy is compatible with Boc- and 9-fluorenylmethoxycarbonyl (Fmoc)-chemistry, allows site-specific labelling of free amines anywhere in the peptide sequence, and can also be used for the introduction of Cy3/Cy5 fluorescence resonance energy transfer (FRET) pairs.

Grant information

We thank Dr Debby Feytens for some of the Cy5 precursor synthesis work and Dr Han-Shen Tae for the Cy5 control experiments. MM was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program (714366), by the Australian Research Council (DE150100784, DP190101667), and by the Vienna Science and Technology Fund (WWTF; LS18–053). PFA, RJL, and DJA were supported by Program Grant APP1072113 from the National Health & Medical Research Council (NHMRC) of Australia and NHMRC Principal Research Fellowships to PFA (APP1080593) and to RJL (APP1072113). Finally, we want to recognise Professor Paul Alewood’s outstanding contributions to the field of peptide science and thank him for his long-lasting collegiality, mentorship, and friendship.

  • Synonyms:
    Cy5.5-PEG-NHS
    Cyanur5.5-PEG-Succinimidyl ester
  • Purity:
    ≥95%
  • Recommended Storage Condition:
    Store at -5°C,keep in dry and avoid sunlight.
  • Uses:
    Applicated in medical research, drug-release, nanotechnology and new materials research, cell culture. In the study of ligand, polypeptide synthesis support, a graft polymer compounds, new materials, and polyethylene glycol-modified functional coatings and other aspects of the active compound.

Cyanine5 Boc-hydrazide, 1 mg

Hersteller Lumiprobe
Kategorie
  • Reagents
Typ Reagents
Specific against other
Menge 1mg
ArtNr LPB-130A0
eClass 6.1 32160401
eClass 9.0 42051003

In-solution conjugation is the most commonly used strategy to label peptides and proteins with fluorophores. However, lack of site-specific control and high costs of fluorophores are recognised limitations of this approach. Here, we established facile access to grams of Cy5-COOH via a two-step synthetic route, demonstrated that Cy5 is stable to HF treatment and therefore compatible with Boc-SPPS, and coupled Cy5 to the N-terminus of α-conotoxin RgIA while still attached to the resin. Folding of the two-disulfide containing Cy5-RgIA benefitted from the hydrophobic nature of Cy5 resulting in only the globular disulfide bond isomer. In contrast, wild-type α-RgIA folded into the inactive ribbon and bioactive globular isomer under the same conditions. Labelled α-RgIA retained its ability to inhibit acetylcholine(100 μM)-evoked current reversibly with an IC50 of 5.0 nM (Hill coefficient = 1.7) for α-RgIA and an IC50 of 1.6 (Hill coefficient = 1.2) for Cy5-RgIA at the α9α10 nicotinic acetylcholine receptors (nAChRs) heterologeously expressed in Xenopus oocytes. Cy5-RgIA was then used to successfully visualise nAChRs in RAW264.7 mouse macrophage cell line. This work introduced not only a new and valuable nAChR probe, but also a new versatile synthetic strategy that facilitates production of milligram to gram quantities of fluorophore-labelled peptides at low cost, which is often required for in vivo experiments. The strategy is compatible with Boc- and Fmoc-chemistry, allows for site-specific labelling of free amines anywhere in the peptide sequence, and can also be used for the introduction of Cy3/Cy5 FRET pairs.

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