与氨基反应的荧光探针.doc

与氨基反应的荧光探针
Highlights of Rhodamine and TRITC Labeling Kits:
· Fast – label and purify antibody in about one hour
· Amine-specific labeling – the NHS-ester or isothiocyanate efficiently labels antibodies and other purified proteins at primary amines (lysine side chains)
· Optimized procedure – following the standard protocol results in antibodies with excellent dye:protein ratios for optimum activity and fluorescence
· Single-use fluors – no need to weigh tiny amounts powder; kits contain single-use vials of reagent
· Efficient purification – kits include purification resin and easy-to-use spin columns, ensuring rapid and efficient removal of non-reacted dye and excellent protein recovery
Applications for Rhodamine Labeling Reagents:
· Label antibodies for use as immunofluorescent probes
· Label oligonucleotides for hybridization probes
· Detect proteins in gels and on Western blots
Properties of Rhodamine Dyes:

Thermo Scientific Pierce Rhodamine Dyes are mixtures of isomers with reactive groups attached at the 5- and 6-positions of the bottom ring. The properties of these isomers are indistinguishable in terms of excitation and emission spectra, and for protein applications there is no need to isolate a specific isomer.
TRITC is the base tetramethylrhodamine molecule functionalized with an isothiocyanate reactive group (–N=C=S) at one of two hydrogen atoms on the bottom ring of the structure. This derivative is reactive towards primary amine groups on proteins, peptides and other biomolecules. NHS-Rhodamine is activated with the N-hydroxy-succinimidyl-ester (NHS ester) functional group. Compared to TRITC, the NHS-ester deriviative has greater specificity toward primary amines in the presence of other nucleophiles and results in a more stable linkage following labeling. Texas Red* Sulfonyl Chloride is a long-wavelength derivative of rhodamine that is modified with sulfonyl chloride for reaction to primary amines. We recommend DyLight 594 NHS Ester as an alternative to Texas Red Sulfonyl Chloride because it is brighter, more photostable and has a more specific labeling chemistry that can be used at physiologic pH.
AMCA (aminomethylcoumarin acetate) is a blue fluorescent dye whose reactive derivatives are used as contrasting probes for double and triple labeling in immunofluorescence microscopy, arrays and in situ hybridization. The desirable properties of AMCA dyes also include a relatively large Stoke's shift and resistance to photobleaching. NHS-AMCA and Sulfo-NHS-AMCA are reactive towards primary amine groups on proteins, peptides and other biomolecules, while AMCA HPDP reacts with sulfhydryls, and AMCA hydrazide is used to label glycosylation sites. AMCA Hydrazide can also be reacted with carboxyl groups using the crosslinker EDC. AMCA has an excitation maximum at 345nm and an emission maximum at 450nm.

NHS-Rhodamine
TRITC
Texas Red* Sulfonyl Chloride
Alternative names
5/6-carboxy-tetramethyl-rhodamine succinimidyl ester, 5/6-TAMRA SE
5(6)-tetramethyl-rhodamine isothiocyanate mixed isomer, 5/6-TRITC, TMR
Sulforhodamine 101 sulfonyl chloride, Texas Red-X, T1905, T353
Molecular weight
527.5
479.0 (chloride salt);
443.5 (no Cl)
625.15
Excitation source
546nm spectral line, mercury-arc lamp; 543nm spectral line, He-Ne laser
546nm spectral line, mercury-arc lamp; 543nm spectral line, He-Ne laser
568nm spectral line, Ar-Kr laser; 594nm spectral line orange He-Ne laser
Excitation wavelength
552nm
544nm
596nm
Emission wavelength
575nm
572nm
615nm
Extinction coefficient
>80,000/M cm
>80000/M cm
~80,000/M cm
CAS #
246256-50-8
95197-95-8
82354-19-6
Purity
> 90% by HPLC
> 85% by HPLC
> 85% by HPLC
Solubility
Soluble in DMF or DMSO
Soluble in DMF or DMSO
Soluble in DMF or methylene chloride (not stable in DMSO)
Storage
Desiccated at -20°C,
protect from moisture,
use only fresh solutions
Desiccated at -20°C,
protect from moisture,
use only fresh solutions
Desiccated at -20°C,
protect from moisture,
use only fresh solutions
Reactive groups
NHS ester, reacts with primary amines at pH 7.0 to 9.0
Isothiocyanate, reacts with primary amines at pH 7.0 to 9.0
Sulfonyl chloride, reacts with primary amines at pH 7.0 to 9.0

与巯基反应的荧光探针
Properties of AMCA Dyes:

NHS-AMCA
Sulfo-NHS-AMCA
AMCA-HPDP
AMCA-Hydrazide
Alternative names
Succinimydyl-7-amino-
4-methylcoumarin-
3-acetic acid,
AMCA NHS Ester,
(like AMCA-X SE)
Sulfosuccinimydyl-
7-amino-4-methyl-
coumarin-3-acetic acid,
AMCA Sulfo-NHS Ester
N-([6-7-amino-
4-methylcoumarin-
3-acetomido] hexyl)-
3'-(2'-pyridyldithio)
proprionamide,
AMCA-pyridyldithiol
Aminomethyl-
coumarin
hydrazide
Molecular weight
330.29
432.34
528.69
247.25
Excitation wavelength
345 to 350nm
345 to 350nm
345 to 350nm
345 to 350nm
Emission wavelength
440 to 460nm
440 to 460nm
440 to 460nm
440 to 460nm
Extinction coefficient
~19,000/M cm
~19,000/M cm
~19,000/M cm
~19,000/M cm
Purity
> 90% by HPLC
> 90% by HPLC
> 95% by HPLC
> 90% by HPLC
Solubility
Soluble in DMF, DMSO
Soluble in water, DMSO
Soluble in DMF
Soluble in DMF
Storage
Desiccated at -20°C,
protect from moisture,
use fresh solutions
Desiccated at -20°C,
protect from moisture,
use fresh solutions
Desiccated at 4°C
Desiccated at 4°C
Reactive groups
NHS ester, reacts
with primary amines
at pH 7.0 to 9.0
Sulfo-NHS ester, reacts
with primary amines
at pH 7.0 to 9.0
Pyridyldithiol, reacts
with sulfhydryls (e.g.,
reduced cysteines)
to form disulfide bonds
Hydrazide, reacts with
aldehydes (oxidized
sugars), or the amine
can be conjugated to carboxyls using EDC.
Related Products and Resources:
DyLight 405 is a newer blue fluorescent dye, tailored for various chemical and biological applications where greater photostability and fluorescence intensity, pH independence, and a narrower emission spectrum are required.
DyLight 405 Antibody Labeling Kits
DyLight 405 and other DyLight Reactive Fluors
Tech Tip #31 – Calculate dye:protein (F/P) molar ratios
Fluorescent Labeling – Top-level menu of all fluorescent reagents
*Trademark, see link in page footer.
和羰基反应的荧光探针（Carbohydrate Reactive Fluorescent Probe）。
Properties of DAPI Fluorescent Dye:
Structure

Alternative names
DAPI Stain, DAPI Dye, DNA Content Counterstain
Chemical name(s)
4,6'-diamidino-2-phenylindole, dihydrochloride
4',6-diamidine-2-phenyl indole
2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride
Molecular formula
C16H15N5 2HCl
Molecular weight
350.25
Excitation wavelength
341 ±3nm near 360nm when bound to dsDNA)
Emission wavelength
452 ±3nm (456 to 460nm when bound to dsDNA)
Extinction coefficient
> 30,600/M cm at 347nm in methanol
CAS #
28718-90-3
Purity
> 95% (most lots >98%) by HPLC at 240nm
Solubility
> 1mg/mL in water; compound is soluble in DMF, water and various non-phosphate aqueous buffers
Storage
Store solid at room temperature (RT), protected from light
Store DAPI solution (1mg/mL) at -20°C protected from light
Reactive groups
None; binds to minor groove of double-stranded DNA