568 nm – VectorLabs

5-ROX (5-Carboxy-X-Rhodamine)

Vector Laboratories R&D — Tue, 19 Sep 2023 22:50:01 +0000

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Description

The carboxylic acid of 5-ROX is used for oligonucleotide labeling and automated DNA sequencing applications. Often a dye of choice for fluorescence resonance energy transfer (FRET) and fluorescence quenching applications. Conjugates of this dye have longer-wavelength spectra than the spectra of Lisaamine™ rhodamine B conjugates, but somewhat shorter-wavelength spectra than those of Texas Red™ conjugates.

Specifications

Unit Size	25 mg, 100 mg, 1000 mg
Reactivity	Primary amines
Abs/Em Maxima	570/591 nm
Extinction coefficient	93,000 cm-1M-1
Solubility	DMSO, DMF, MeOH
Spectrally similar dyes	Alexa Fluor® 568, TAMRA, CF™ 568
CAS	216699-35-3
Molecular weight	534.61
Storage Conditions	-20°C.
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Protein Labeling Calculator

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Protein molecular weight (daltons)

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Volume of protein to be labeled (µL)

Enter only one — either volume or amount of protein to be labeled

Amount of protein to be labeled (mg)

Molecular weight of labeling reagent (Da)

Amount of labeling reagent used (mg)

Maximum concentration of DMSO (%) Leave blank or 0 if not used

Enter only one — either maximum concentration of DMSO or volume DMSO added to label

Volume of anhydrous DMSO added (µL)

Molar equivalents of labeling reagent used

Volume of DMSO required to make a stock solution (µL)

Volume of protein to be labeled (µL)

Amount of stock labeling reagent to be added (µL)

Amount of DMSO in labeling reaction (%)

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5-ROX NHS Ester

Vector Laboratories R&D — Tue, 19 Sep 2023 22:49:59 +0000

Description
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Abs/Em Spectra
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Description

5-Carboxy-X-rhodamine N-succinimidyl ester (5-ROX NHS Ester) is used as an amine coupling reagent to form 5-carboxy-X-rhodamine (ROX) derivatized compounds such as proteins, nucleic acids and drugs that may be analyzed by fluorescence resonance energy transfer (FRET) and fluorescence quenching applications. Labeling reagent for preparation of charge-modified dye-labeled ddNTPs to “direct-load” DNA sequencing. Conjugates of this dye have longer-wavelength spectra than the spectra of Lisaamine™ rhodamine B conjugates, but somewhat shorter-wavelength spectra than those of Texas Red™ conjugates.

Specifications

Unit Size	25 mg, 100 mg, 1000 mg
Reactivity	Primary amines
Abs/Em Maxima	582/606 nm
Extinction coefficient	93,000 cm-1M-1
Solubility	DMSO, DMF, MeOH
Spectrally similar dyes	Alexa Fluor® 568, TAMRA, CF™ 568
CAS	209734-74-7
Molecular weight	631.69
Storage Conditions	-20°C.
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Protein Labeling Calculator

Protein name

Protein molecular weight (daltons)

Protein concentration (mg/mL)

Volume of protein to be labeled (µL)

Enter only one — either volume or amount of protein to be labeled

Amount of protein to be labeled (mg)

Molecular weight of labeling reagent (Da)

Amount of labeling reagent used (mg)

Maximum concentration of DMSO (%) Leave blank or 0 if not used

Enter only one — either maximum concentration of DMSO or volume DMSO added to label

Volume of anhydrous DMSO added (µL)

Molar equivalents of labeling reagent used

Volume of DMSO required to make a stock solution (µL)

Volume of protein to be labeled (µL)

Amount of stock labeling reagent to be added (µL)

Amount of DMSO in labeling reaction (%)

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AZDye 568 Cadaverine

Vector Laboratories R&D — Tue, 19 Sep 2023 22:47:32 +0000

Description
Specifications
Abs/Em Spectra
Documents

Description

AZDye™ 568 Cadaverine is a carbonyl reactive building block used to modify carboxylic groups in the presence of activators (e.g. EDC, or DCC) or activated esters (e.g. NHS esters) through a stable amide bond. It also can be employed as a polar tracer and as a reactive dye for labeling proteins via a carboxylic acid moiety.

AZDye™ 568 is a bright, and highly photostable, orange-fluorescent probe optimally excited by the 568 nm laser line on the Ar-Kr mixed-gas laser. This probe is water-soluble and its fluorescence is pH independent over a wide pH range. The brightness and photostability of this dye are best suited to direct imaging of low-abundance targets.

Specifications

Unit Size	1 mg, 5 mg, 25 mg, 100 mg
Reactivity	Aldehyde, carboxylic acid, ketone
Abs/Em Maxima	578/602 nm
Extinction coefficient	93,000 cm-1M-1
Solubility	Water, DMSO, DMF
Spectrally similar dyes	Alexa Fluor® 568, CF™ 568
Molecular weight	892.92 (TFA salt)
Storage Conditions	-20°C.
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Documents

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AZDye 568 Hydroxylamine

Vector Laboratories R&D — Tue, 19 Sep 2023 22:47:31 +0000

Description
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Abs/Em Spectra
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Description

AZDye 488 Hydroxylamine (aminooxy) reacts with aldehydes and ketones to yield oximes. Oximes are superior to hydrazones with respect to hydrolytic stability. AZDye 568 Hydroxlamine often used as membrane-impermeant, aldehyde-fixable cell tracers as well as reactive dye for labeling aldehydes or ketones in polysaccharides or glycoproteins.

Specifications

Unit Size	1 mg, 5 mg, 25 mg
Reactivity	Aldehyde, ketone
Abs/Em Maxima	578/602 nm
Extinction coefficient	93,000 cm-1M-1
Solubility	Water, DMSO, DMF
Spectrally similar dyes	Alexa Fluor® 568, CF™ 568
Molecular weight	766.84 (protonated)
Storage Conditions	-20°C.
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Protein Labeling Calculator

Protein name

Protein molecular weight (daltons)

Protein concentration (mg/mL)

Volume of protein to be labeled (µL)

Enter only one — either volume or amount of protein to be labeled

Amount of protein to be labeled (mg)

Molecular weight of labeling reagent (Da)

Amount of labeling reagent used (mg)

Maximum concentration of DMSO (%) Leave blank or 0 if not used

Enter only one — either maximum concentration of DMSO or volume DMSO added to label

Volume of anhydrous DMSO added (µL)

Molar equivalents of labeling reagent used

Volume of DMSO required to make a stock solution (µL)

Volume of protein to be labeled (µL)

Amount of stock labeling reagent to be added (µL)

Amount of DMSO in labeling reaction (%)

Documents

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AZDye 568 Maleimide

Vector Laboratories R&D — Tue, 19 Sep 2023 22:39:11 +0000

Description
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Abs/Em Spectra
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Description

AZDye™ 568 Maleimide is a bright, and highly photostable, orange-fluorescent probe optimally excited by the 568 nm laser line on the Ar-Kr mixed-gas laser. AZDye™ 568 Maleimide is water-soluble and its fluorescence is pH independent over a wide pH range. The brightness and photostability of this dye are best suited to direct imaging of low-abundance targets.

Maleimide is the most popular sulfhydryl-reactive reactive group for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The maleimide group specifically and efficiently reacts with reduced thiols (sulfhydryl groups, –SH) at pH 6.5 to 7.5 to form stable thioether bond. The resulting conjugates exhibit brighter fluorescence and greater photostability than the conjugates of many other spectrally similar fluorophores.

Specifications

Unit Size	1 mg, 5 mg, 25 mg, 100 mg
Reactivity	Primary amine
Abs/Em Maxima	575/600 nm
Extinction coefficient	92,000 cm-1M-1
Solubility	Water, DMSO, DMF
Spectrally similar dyes	Alexa Fluor® 568, CF™ 568
Molecular weight	857.93
Storage Conditions	-20°C.
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Protein Labeling Calculator

Protein name

Protein molecular weight (daltons)

Protein concentration (mg/mL)

Volume of protein to be labeled (µL)

Enter only one — either volume or amount of protein to be labeled

Amount of protein to be labeled (mg)

Molecular weight of labeling reagent (Da)

Amount of labeling reagent used (mg)

Maximum concentration of DMSO (%) Leave blank or 0 if not used

Enter only one — either maximum concentration of DMSO or volume DMSO added to label

Volume of anhydrous DMSO added (µL)

Molar equivalents of labeling reagent used

Volume of DMSO required to make a stock solution (µL)

Volume of protein to be labeled (µL)

Amount of stock labeling reagent to be added (µL)

Amount of DMSO in labeling reaction (%)

Documents

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AZDye 568 NHS Ester

Vector Laboratories R&D — Tue, 19 Sep 2023 22:38:44 +0000

Description
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Abs/Em Spectra
Protein Labeling Calculator
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Description

AZDye™ 568 NHS Ester (Alexa Fluor 568 NHS Ester equivalent) is an amine-reactive, orange fluorescent dye routinely used to label proteins or antibodies through the primary amines (Lys), amine-modified oligonucleotides, and other amine-containing biomolecules. AZDye™ 568 dye can be used for proteins labeling at high molar ratios without significantself-quenching, enabling brighter conjugates and more sensitive detection.

AZDye™ 568 is a water-soluble, photostable, and bright orange dye with pH insensitive emission from pH 4 to pH 10. The excitation of AZDye™ 568 dye ideally suited for the 568 nm laser line on the Ar-Kr mixed-gas laser. AF™ 568 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates often used for generation of stable signal in imaging and flow cytometry.

Specifications

Unit Size	1 mg, 5 mg, 25 mg, 100 mg
Reactivity	Primary amine
Abs/Em Maxima	578/602 nm
Extinction coefficient	88,000 cm-1M-1
Solubility	Water, DMSO, DMF
Spectrally similar dyes	Alexa Fluor® 568, CF™ 568
Molecular weight	791.80 (protonated)
Storage Conditions	-20°C.
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Protein Labeling Calculator

Protein name

Protein molecular weight (daltons)

Protein concentration (mg/mL)

Volume of protein to be labeled (µL)

Enter only one — either volume or amount of protein to be labeled

Amount of protein to be labeled (mg)

Molecular weight of labeling reagent (Da)

Amount of labeling reagent used (mg)

Maximum concentration of DMSO (%) Leave blank or 0 if not used

Enter only one — either maximum concentration of DMSO or volume DMSO added to label

Volume of anhydrous DMSO added (µL)

Molar equivalents of labeling reagent used

Volume of DMSO required to make a stock solution (µL)

Volume of protein to be labeled (µL)

Amount of stock labeling reagent to be added (µL)

Amount of DMSO in labeling reaction (%)

Documents

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AZDye 568 TFP Ester

Vector Laboratories R&D — Tue, 19 Sep 2023 22:38:42 +0000

Description
Specifications
Abs/Em Spectra
Protein Labeling Calculator
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Description

AZDye™ 568 TFP Ester is an amine-reactive, orange fluorescent dye routinely used to label proteins or antibodies through the primary amines (Lys), amine-modified oligonucleotides, and other amine-containing biomolecules. AZDye™ 568 dye can be used for proteins labeling at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection.

TFP (tetrafluorophenyl) ester is an amine-reactive activated ester that reacts with primary amines of biomolecules in the same way as the succinimidyl ester (SE or NHS-ester) to form a stable amide bond. The major advantage of TFP esters over the succinimidyl ester is much improved resistance to spontaneous hydrolysis during conjugation reactions resulting in more efficiency and better reproducible labeling of biopolymers. TFP esters are stable for several hours at the basic pH typically used for reactions–far outlasting succinimidyl esters.

Specifications

Unit Size	1 mg, 5 mg, 25 mg
Reactivity	Primary amine
Abs/Em Maxima	578/602 nm
Extinction coefficient	88,000 cm-1M-1
Solubility	Water, DMSO, DMF
Spectrally similar dyes	Alexa Fluor® 568, CF™ 568
Molecular weight	842.79 (protonated)
Storage Conditions	-20°C.
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Protein Labeling Calculator

Protein name

Protein molecular weight (daltons)

Protein concentration (mg/mL)

Volume of protein to be labeled (µL)

Enter only one — either volume or amount of protein to be labeled

Amount of protein to be labeled (mg)

Molecular weight of labeling reagent (Da)

Amount of labeling reagent used (mg)

Maximum concentration of DMSO (%) Leave blank or 0 if not used

Enter only one — either maximum concentration of DMSO or volume DMSO added to label

Volume of anhydrous DMSO added (µL)

Molar equivalents of labeling reagent used

Volume of DMSO required to make a stock solution (µL)

Volume of protein to be labeled (µL)

Amount of stock labeling reagent to be added (µL)

Amount of DMSO in labeling reaction (%)

Documents

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AZDye 568 Tyramide

Vector Laboratories R&D — Tue, 19 Sep 2023 20:09:42 +0000

Description
Specifications
Abs/Em Spectra
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FAQs

Description

Red fluorescent reagent widely used for tyramide signal amplification (TSA) in IHC, ICC, FISH and multicolor FISH. Tyramide signal amplification (TSA) has proven to be a particularly versatile and powerful enzyme amplification technique with improved assay sensitivity. TSA is based on the ability of HRP, in the presence of low concentrations of hydrogen peroxide, to convert labeled tyramine-containing substrate into an oxidized, highly reactive free radical that can covalently bind to tyrosine residues at or near the HRP.

AZDye™ 568 Tyramide is a component of many tyramide signal amplification kits, including SuperBoost®. This reagent can be used with any antibody conjugated to HRP for tyramide signal amplification for detection of low abundance targets in multiplexable fluorescent immunocytochemistry (ICC), immunohistochemistry (IHC ), and in situ hybridization (ISH).

Specifications

Unit Size	150 slides, 1500 slides
Abs/Em Maxima	578/602 nm
Extinction Coefficient	88,000
Flow Cytometry Laser Line	532 nm or 568 nm
Microscopy Laser Line	532 nm or 568 nm
Spectrally Similar Dyes	Alexa Fluor® 568, CF® 568
Molecular weight	813.89
CAS	N/A
Solubility	Water, DMSO, DMF
Appearance	Dark blue solid
Storage Conditions	-20°C. Desiccate
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Documents

FAQs

What is the quantity of tyramide reagent provided in the 150 slides and 1500 slides unit sizes?

150 slides has 0.24 µmol (depending on molecular weight this is ~200 µg)

1500 slides has 2.4 µmol (depending on molecular weight this is ~2 mg)

What is the protocol for using a Fluorophore Tyramide product in tyramide signal amplification (TSA)?

Here is a general protocol for the 150 slide unit size. To prepare 100x stock solution, we recommend reconstituting with 150 ul of DMSO. Storage once in solution: 100X stock solution in DMSO can be stored for 6 months at 2-8 °C protected from light. Avoid moisture.

Working solutions can be made fresh by adding 1 part 100X stock solution, 1 part 0.15% H202, and 100 part buffer.

For example, you can add 5 µL of 100X Tyramide stock solution and 5 µL 0.15% H2O2 to 500 µL of buffer. The final concentration of H2O2 is 0.0015%.

Typical reaction time following the HRP reagent is 2-10 minutes but should be optimized for each workflow. Following the TSA working solution incubation, wash thoroughly.

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AZDye 568 Azide Plus

Vector Laboratories R&D — Tue, 19 Sep 2023 19:56:10 +0000

Description
Specifications
Abs/Em Spectra
Documents
Click Reaction Protocol

Description

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

Unit Size	1 mg, 5 mg, 25 mg
Abs/Em Maxima	578/602 nm
Extinction Coefficient	88,000
Flow Cytometry Laser Line	532 nm or 568 nm
Microscopy Laser Line	532 nm or 568 nm
Spectrally Similar Dyes	Alexa Fluor® 568, CF® 568
Molecular weight	915.01 (protonated)
CAS	N/A
Solubility	Water, DMSO, DMF
Purity	>95% (HPLC)
Appearance	Red solid
Storage Conditions	-20°C. Desiccate
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Documents

Click Reaction Protocol for Staining Fixed/Permeabilized Cell

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.

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

1 mM Azide Plus reagent in DMSO or water

Table 1

Reaction Component	Number of coverslipsor wells of a 96-well plate
Reaction Component	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

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

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
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)
Add 10 µL of 100 mM THPTA solution, vortex briefly to mix.
Add 10 µL of 20 mM CuSO₄ solution, vortex briefly to mix.
Add 10 µL of 300 mM sodium ascorbate solution to initiate click reaction, vortex briefly to mix.
Vortex continuously or rotate end-over-end for 30 minutes at room temperature.
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.
Centrifuge for 10 minutes at 13,000-20,000 x g, then carefully remove upper aqueous layer without disturbing the interface layer containing proteins.
Add 450 µL of methanol, vortex briefly.
Centrifuge for 5 minutes at 13,000-20,000 × g to pellet protein. Carefully remove and discard supernatant.
Open the lid to microfuge tube and allow protein pellet to air dry. Do not over dry the pellet!

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AZDye 568 Tetrazine

Vector Laboratories R&D — Tue, 19 Sep 2023 19:50:54 +0000

Description
Specifications
Abs/Em Spectra
Documents

Description

A bright, red-fluorescent probe used for detection TCO-tagged biopolymers. AZDye 568 Tetrazine demonstrates exceptionally fast cycloaddition kinetics (up to 30 000 M^-1 s^-1) with trans-cyclooctenes (TCO) as the dienophile, the fastest kinetics ever reported for any bioorthogonal reaction. In applications such as in vivo cancer imaging or pre‐targeted cell labeling studies where rapid reaction kinetics is a must AZDye 568 Tetrazine probe would of great value.

Tetrazines demonstrate exceptionally fast cycloaddition kinetics (up to 30 000 M^-1 s^-1) with trans-cyclooctenes (TCO) as the dienophile, the fastest kinetics ever reported for any bioorthogonal reaction. In addition, inverse-Electron-Demand Diels-Alder reaction of tetrazines trans-cyclooctenea forms a stable covalent bond and does not require Cu-catalyst or elevated temperatures.

AZDye 568™ is a bright, and highly photostable, orange-fluorescent probe optimally excited by the 568 nm laser line on the Ar-Kr mixed-gas laser. This probe is water-soluble and its fluorescence is pH independent over a wide pH range. The brightness and photostability of this dye are best suited to direct imaging of low-abundance targets.

Specifications

Unit Size	1 mg, 5 mg, 25 mg
Abs/Em Maxima	578/602 nm
Extinction Coefficient	88,000
Spectrally Similar Dyes	Alexa Fluor® 568, CF® 488 Dye
Molecular weight	863.92 (protonated)
Solubility	Water, MeOH, DMSO, DMF
Appearance	Red solid
Storage Conditions	-20°C. Desiccate
Shipping Conditions	Ambient temperature

Abs/Em Spectra

Documents

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