The post Difluorocarboxyfluorescein Azide, 6-isomer appeared first on VectorLabs.

SY-21 Azide is a nonfluorescent acceptor dye often used for preparation of peptide and oligonucleotide FRET probes. A broad and intense quenching range from 580 nm to 680 nm makes it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications in conjunction with fluorescent dyes at 580 nm to 680 nm, such as Fluorescein, Alexa Fluor® 568, 594, 633, 647, TAMRA, ROX, Texas Red and Cy5.

SY-21 Azide can be reacted with terminal alkynes via a copper-catalyzed click reaction (CuAAC). It also reacts with strained cyclooctyne via a copper-free “click chemistry” reaction to form a stable triazole and does not require Cu-catalyst or elevated temperatures.

[caption id="attachment_27198" align="alignnone" width="1000"] Abs/Em Spectra[/caption]

Specifications

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SY-9 is a more hydrophilic version of the popular SY-7 dark quencher. This nonfluorescent acceptor dye is often used for the preparation of peptide FRET probes. A broad and intense quenching range from 500-600 nm makes it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications in conjunction with dyes emitting at 500 nm to 600 nm, such as Fluorescein, Oregon Green, Carboxyrhodamine 110 (Rhodamine Green), Alexa Fluor® 488, 532, 546, 555, 568, Cy3, TAMRA and ROX dyes.

SY-9 Azide can be reacted with terminal alkynes via a copper-catalyzed click reaction (CuAAC). It also reacts with strained cyclooctyne via a copper-free “click chemistry” reaction to form a stable triazole and does not require Cu-catalyst or elevated temperatures.

[caption id="attachment_27189" align="alignnone" width="1000"] Abs/Em Spectra[/caption]

Specifications

The post SY-9 Azide appeared first on VectorLabs.

SY-7 Azide is a nonfluorescent acceptor dye used for preparation of peptide and oligonucleotide FRET probes. A broad and intense quenching range from 500-600 nm makes it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications in conjunction with fluorescent dyes at 520 nm to 600 nm, such as Fluorescein, Oregon Green, Carboxyrhodamine 110 (Rhodamine Green), Alexa Fluor® 488, 532, 546, 555, 568, Cy3, TAMRA and ROX dyes.

SY-7 Azide can be reacted with terminal alkynes via a copper-catalyzed click reaction (CuAAC). It also reacts with strained cyclooctyne via a copper-free “click chemistry” reaction to form a stable triazole and does not require Cu-catalyst or elevated temperatures.

[caption id="attachment_27189" align="alignnone" width="1000"] Abs/Em Spectra[/caption]

Specifications

The post SY-7 Azide appeared first on VectorLabs.

Iodoacetamide Azide is a broad spectrum cysteine reactive probe that reacts with nucleophiles, such as cysteine in proteins and peptides to covalently bind cysteine residues into azide group. The azide group can be used as a click-chemistry handle to attach a fluorophore or biotin to reactive cysteines in proteins for visualization by in-gel fluorescence or identification by mass spectrometry, respectively.

Specifications

The post Iodoacetamide Azide appeared first on VectorLabs.

Cyanine heptamethine dyes are well-known NIR fluorophores, with emission wavelengths >740 nm, and often are reagents of choice for labeling antibodies. Two of the most popular commercial NIR cyanine heptamethine dyes for antibody conjugation are IRDye 800CW and DyLight 800. While these NIR dyes are undoubtedly useful for many types of immunofluorescence technologies, the resulting NIR dye-labeled antibodies sometimes exhibit performance limitations due to three inherent fluorophore concerns. (1) A meso-OAryl group connected directly to the heptamethine fluorochrome group is susceptible to nucleophilic displacement by biological amines and thiols^{1, 2} resulting in a diminished chemical stability of the dye-antibody conjugates during synthesis, storage, or the time-course of an imaging experiment. In addition, the electron-donating meso-OAryl group promotes high fluorochrome reactivity with electrophilic singlet oxygen, resulting in relatively poor dye photostability.^3,4 (2) Both dyes are flat molecules with a hydrophobic core and a polyanionic charge periphery. Thus, the chemical conversion of a small polar, cationic lysine residue on the antibody surface to a large hydrophobic, polyanionic dye derivative has the potential to produce substantial changes in antibody folding and physiochemical properties, leading to lower antibody stability and decreased target specificity.^4-8 (3) When activated versions of these hydrophobic dyes are conjugated to an antibody surface, they tend to attach at the proximal lysine sites as stacked face-to-face dimers, which produces a diagnostic H-dimer peak in the absorbance spectra that is nonfluorescent.^8,9 Moreover, the close stacking of proximal conjugated IRDye 800CW or DyLight800 on an antibody surface amplifies the potential for a deleterious effect on antibody targeting because of a localized patch of polyanionic charge and hydrophobicity.

In order to address these drawbacks of commercially NIR cyanine a conceptually new class of sterically shielded NIR dyes was developed in Dr. Bradley D. Smith laboratory.^{10, 11} These dyes contain two shielding PEG arms directly over both faces of the heptamethine fluorochrome blocking any undesired bimolecular association processes and thus enhance the fluorescence brightness. A recently published antibody labeling study clearly demonstrated 1-2 order of magnitude increase in brightness compared to commercially available NIR dyes as a result of almost complete prevention of stacking of multiple fluorophores appended to the antibody surface.^{10, 11}

These sterically shielded NIR dyes with greatly improved chemical and photochemical stability and substantially enhanced brightness will enable researchers to greatly improve various types of indirect NIR immunofluorescence imaging and diagnostics applications that require high sensitivity and also develop new photonintense techniques that require high photostability.

MB 800Z is a zwiteroinic, charge-balanced dye with an equal number of anionic sulfonate and cationic ammonium residues, a structural feature that is known to reduce interactions with off -target biological surfaces.

[caption id="attachment_27084" align="alignnone" width="1000"] Abs/Em Spectra[/caption]

Specifications

The post AZDye 800 Azide appeared first on VectorLabs.

AZDye™ 633 Azide is a bright and photostable far-red fluorescent probe with excitation ideally suited to the 633 nm or 635 nm laser excitation source. AZDye™ 633 Azide is water-soluble, pH-insensitive from pH 4 to pH 10. AZDye™ 633 spectrally is almost identical to Alexa Fluor® 633, DyLight® 633 or CF® 633 Dye. Combination of superior brightness and very low autofluorescence background signal in most biological samples in far-red spectral region allows for very sensitive detection of alkyne-labeled biomolecules.

[caption id="attachment_27033" align="alignnone" width="1000"] Abs/Em Spectra[/caption]

Specifications

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Azide Plus reagents is the most recent step in improving CuAAC reaction in complex media developed by scientists at Click Chemistry Tools. Azide Plus reagents contain a complete copper-chelating system in their structure, allowing for the formation of strong, active copper complexes that act simultaneously as both reactant and catalyst in the CuAAC reaction. This azide-copper complex reacts almost instantaneously with alkynes under diluted conditions. This unprecedented reactivity in the CuAAC reaction is of special value for the detection of low abundance targets, improving biocompatibility, and is also valuable for any other application where greatly improved S/N ratio is highly desired.

Specifications

Abs/Em Spectra

Protocol

This is a general protocol for fixed/permeabilized cell imaging through a copper-catalyzed click reaction using the fluorescent Azide Plus reagent. We recommend using this protocol as a starting point for optimization of particular click chemistry procedures. We have found that a 1.5-3.0 μM concentration of Azide Plus reagent was optimal for most applications, including imaging of EdU incorporated into newly synthesized DNA and imaging of OPP labeled proteins without causing a high background signal. The optimal final concentration of the Azide Plus reagent is sample dependent and may range from 0.5 μM to 10 μM. Final concentrations below or above this range are also possible, and should be optimized per the specific application.

1. Prepare the following click solutions:
   — 50 mM copper sulfate in water
   — 300 mM sodium ascorbate in water (dissolve 60 mg of sodium ascorbate in 1 mL of water)
   — Subtext
2. 1 mM Azide Plus reagent in DMSO or water

   Table 1

   Reac­tion Compo­nent	Number of cover­slipsor wells of a 96-well plate
   	1 cover­slip or 10 wells	5 cover­slips or 50 wells	10 cover­slips or 100 wells	20 cover­slips or 200 wells
   Reaction Buffer (Tris)	888 µL	4.4 mL	8.9 mL	17.8 mL
   50 mM Copper Sulfate	10 µL	50 µL	100 µL	200 µL
   AZDye Azide Plus­ Solu­tion (2 µM final concentration)	2 µL	10 µL	20 µL	40 µL
   Sodium ascorbate	100 µL	500 µL	1 mL	2 mL
   Total Volume	1 mL	5 mL	10 mL	20 mL

3. Remove the permeabilization buffer (if used). Wash the cells in each well twice with 1 ml of PBS. Remove PBS.
4.  Immediately add 1 mL of the Reaction Cocktail to the sample. Evenly distribute the reaction cocktail over the sample.
5. Protect from light, and incubate the plate for 30 minutes at room temperature.
6. 6Remove the reaction cocktail. Wash each well once with 1 ml of Wash Buffer. Remove the Wash Buffer.
7. Wash each well with 1 mL of PBS. Remove PBS.

Click Reaction Protocol for Cell Lysates Labeling

This is a general protocol for labeling proteins in cell lysate through a copper-catalyzed click reaction using the fluorescent Azide Plus reagent. We recommend using this protocol as a starting point for optimization of particular click chemistry procedures. We have found that a 20 μM concentration of Azide Plus reagent was sufficient to label all alkyne-tagged proteins in the cell lysate without causing a high background signal. The optimal final concentration of the Azide Plus reagent is sample dependent and may range from 5 μM to 50 μM. Final concentrations below or above this range are also possible, and should be optimized per the specific application.

1. Prepare the following click solutions:
   — 100 mM THPTA ligand in water (100 mg of THPTA in 2.3 mL of water)
   — 20 mM copper sulfate in water (dissolve 11.6 mg of copper II sulfate pentahydrate in 2.3 mL of water)
   — 300 mM sodium ascorbate in water (dissolve 60 mg of sodium ascorbate in 1 mL of water)
   — 1 mM Azide Plus reagent in DMSO or water
2. For each protein lysate sample, add the following to a 1.5 mL microfuge tube, then vortex briefly to mix.
   — 50 µL of protein lysate (1-5 mg/mL) in protein extraction buffer
   — 120 µL of Tris buffer
   — 4 µL of Azide Plus reagent stock solution (5 μM final concentration)
3. Add 10 µL of 100 mM THPTA solution, vortex briefly to mix.
4. Add 10 µL of 20 mM CuSO₄ solution, vortex briefly to mix.
5. Add 10 µL of 300 mM sodium ascorbate solution to initiate click reaction, vortex briefly to mix.
6. Vortex continuously or rotate end-over-end for 30 minutes at room temperature.
7. Add the labeling reaction to 3 mL of cold (–20°C) methanol, 0.75 ml of Chloroform and 2.1 mL of water. Cool it to –20°C for 1 hour.
8. Centrifuge for 10 minutes at 13,000-20,000 x g, then carefully remove upper aqueous layer without disturbing the interface layer containing proteins.
9. Add 450 µL of methanol, vortex briefly.
10. Centrifuge for 5 minutes at 13,000-20,000 × g to pellet protein. Carefully remove and discard supernatant.
11. Open the lid to microfuge tube and allow protein pellet to air dry. Do not over dry the pellet!

The post AZDye 680 Azide Plus appeared first on VectorLabs.

AZDye 680 is a bright and photostable near-IR dye that is spectrally identical to Alexa Fluor® 680 and IRDye® 680RD. AZDye 680 is often used for small animal imaging applications in the 700 nm wavelength and provides excellent clearance profiles in animals. Flow cytometry is another common application for this dye. AZDye 680 ideally suited for the 633 nm laser line.

Alexa Fluor® is a registered trademark of Thermo Fisher Scientific.

[caption id="attachment_24263" align="alignnone" width="1940"] Abs/Em Spectra[/caption]

Specifications

The post AZDye 680 Azide appeared first on VectorLabs.

Azide Plus reagents is the most recent step in improving CuAAC reaction in complex media developed by scientists at Click Chemistry Tools. Azide Plus reagents contain a complete copper-chelating system in their structure, allowing for the formation of strong, active copper complexes that act simultaneously as both reactant and catalyst in the CuAAC reaction. This azide-copper complex reacts almost instantaneously with alkynes under diluted conditions. This unprecedented reactivity in the CuAAC reaction is of special value for the detection of low abundance targets, improving biocompatibility, and is also valuable for any other application where greatly improved S/N ratio is highly desired.

[caption id="attachment_24144" align="alignnone" width="1000"] Abs/Em Spectra[/caption]

Specifications

The post TAMRA Azide Plus appeared first on VectorLabs.