与氨基反应的荧光探针

1、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 hourAmine-specific labeling - the NHS-ester or isothiocyanate efficiently labels antibodies and other purified proteins at primary amines (lysine side chains)Optimized procedure - following the st

2、andard protocol results in antibodies with excellent dye:protein ratios for optimum activity and fluorescenceSingle-use fluors - no need to weigh tiny amounts powder; kits contain single-use vials of reagentEfficient purification - kits include purification resin and easy-to-use spin columns, ensuri

3、ng rapid and efficient removal of non-reacted dye and excellent protein recoveryApplications for Rhodamine Labeling Reagents:Label antibodies for use as immunofluorescent probesLabel oligonucleotides for hybridization probesDetect proteins in gels and on Western blotsProperties of Rhodamine Dyes:The

4、rmo 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 speci

5、fic 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-Rhodam

6、ine 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

7、 is a long-wavelength derivative of rhodamine that is modified with sulfonyl chloride for reaction to primary amines. We recommend DyLight594 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 use

8、d 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 relativel

9、y large Stokes 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 reac

10、ted with carboxyl groups using the crosslinker EDC. AMCA has an excitation maximum at 345nm and an emission maximum at 450nm.NHS-RhodamineTRITCTexas Red* Sulfonyl ChlorideAlternative names5/6-carboxy-tetramethyl-rhodamine succinimidyl ester, 5/6-TAMRA SE5(6)-tetramethyl-rhodamine isothiocyanate mixe

11、d isomer, 5/6-TRITC, TMRSulforhodamine 101 sulfonyl chloride, Texas Red-X, T1905, T353Molecular weight527.5479.0 (chloride salt);443.5 (no Cl)625.15Excitation source546nm spectral line, mercury-arc lamp; 543nm spectral line, He-Ne laser546nm spectral line, mercury-arc lamp; 543nm spectral line, He-N

12、e laser568nm spectral line, Ar-Kr laser; 594nm spectral line orange He-Ne laserExcitation wavelength552nm544nm596nmEmission wavelength575nm572nm615nmExtinction coefficient80,000/M cm80,000/M cm80,000/M cmCAS #246256-50-895197-95-882354-19-6Purity 90% by HPLC 85% by HPLC 85% by HPLCSolubilitySoluble

13、in DMF or DMSOSoluble in DMF or DMSOSoluble in DMF or methylene chloride (not stable in DMSO)StorageDesiccated at -20C, protect from moisture, use only fresh solutionsDesiccated at -20C, protect from moisture, use only fresh solutionsDesiccated at -20C, protect from moisture, use only freshsolutions

14、Reactive groupsNHS ester, reacts with primary amines at pH 7.0 to 9.0Isothiocyanate, reacts with primary amines at pH 7.0 to 9.0Sulfonyl 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-AMCASulfo-NHS-AMCAAMCA-HPDPAMCA-HydrazideAlternative namesSuccinimydyl-7

15、-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 EsterN-(6-7-amino- 4-methylcoumarin- 3-acetomido hexyl)- 3-(2-pyridyldithio) proprionamide, AMCA-pyridyldithiolAminomethylcoumarin hydrazideMolecular we

16、ight330.29432.34528.69247.25Excitation wavelength345 to 350nm345 to 350nm345 to 350nm345 to 350nmEmission wavelength440 to 460nm440 to 460nm440 to 460nm440 to 460nmExtinction coefficient19,000/M cm19,000/M cm19,000/M cm19,000/M cmPurity 90% by HPLC 90% by HPLC 95% by HPLC 90% by HPLCSolubilitySolubl

17、e in DMF, DMSOSoluble in water, DMSOSoluble in DMFSoluble in DMFStorageDesiccated at -20C, protect from moisture, use fresh solutionsDesiccated at -20C, protect from moisture, use fresh solutionsDesiccated at 4CDesiccated at 4CReactive groupsNHS ester, reacts with primary amines at pH 7.0 to 9.0Sulf

18、o-NHS ester, reacts with primary amines at pH 7.0 to 9.0Pyridyldithiol, reacts with sulfhydryls (e.g., reduced cysteines) to form disulfide bondsHydrazide, reacts with aldehydes (oxidized sugars), or the amine can be conjugated to carboxyls using EDC.Related Products and Resources:DyLight 405 is a n

19、ewer 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 KitsDyLight 405 and other DyLight Reactive FluorsTech Tip #31 - C

20、alculate dye:protein (F/P) molar ratiosFluorescent 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:StructureCl a s ClKH;玲 名H:DAPI B-Dian no-2-phenylindo c d hydnocloride MW35D.25Alternative namesDAPI Stain, DAPI Dye, DNA Content CounterstainChemical name(s)4,6-diamidino-2-phenylindol