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Storage Temp | Protected from light,Store at -20°C |
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Shipped In | Ice chest + Ice pads |
Product Description | Product introduction: Alexa Fluor™ 555 is a bright orange dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor™ 555 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor™ 555 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more). The maleimide derivative of Alexa Fluor™ 555 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor™ 555 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores. Detailed information about this AlexaFluor™ maleimide: Fluorophore label: Alexa Fluor™ 555 dye Reactive group: maleimide Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates Ex/Em of the conjugate: 556/572 nm Extinction coefficient: 158,000 cm-1M-1 Spectrally similar dyes: Tetramethylrhodamine Molecular weight: ∼1250 Typical Conjugation Reaction The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation. The Alexa Fluor™ maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol. Conjugate Purification Labeled antibodies are typically separated from free Alexa Fluor™ dye using a gel filtration column, such as Sephadex™ G-25, BioGel™ P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate: Antibody Conjugate Purification Kit for 0.5-1 mg () Antibody Conjugate Purification Kit for 20-50 µg () Antibody Conjugate Purification kit for 50-100 µg () Learn More About Protein and Antibody Labeling We offer a wide selection of Molecular Probes™ antibody and protein labeling kits to fit your starting material and your experimental setup. See ourAntibody Labeling kits or use ourKits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes™ Handbook. We’ll Make a Custom Conjugate for You If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Ourcustom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified. |
Reactivity | Thiol |
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1. Smiley RD, Zhuang Z, Benkovic SJ, Hammes GG. () Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation.. Biochemistry, [PMID:16800624] |
2. McGeoch AT, Trakselis MA, Laskey RA, Bell SD. () Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism.. Nat Struct Mol Biol, [PMID:16116441] |
3. Antikainen NM, Smiley RD, Benkovic SJ, Hammes GG. () Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase.. Biochemistry, [PMID:16363797] |
4. Zhang Z, Spiering MM, Trakselis MA, Ishmael FT, Xi J, Benkovic SJ, Hammes GG. () Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies.. Proc Natl Acad Sci U S A, [PMID:15728347] |
5. Weninger K, Bowen ME, Chu S, Brunger AT. () Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations.. Proc Natl Acad Sci U S A, [PMID:14657376] |
6. Yang W, Musser SM. () Nuclear import time and transport efficiency depend on importin beta concentration.. J Cell Biol, [PMID:16982803] |
7. DeRocco V, Anderson T, Piehler J, Erie DA, Weninger K,. () Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.. Biotechniques, [PMID:21091445] |
8. McCann JJ, Choi UB, Zheng L, Weninger K, Bowen ME,. () Optimizing methods to recover absolute FRET efficiency from immobilized single molecules.. Biophys J, [PMID:20682275] |
9. Knight JD, Falke JJ,. () Single-molecule fluorescence studies of a PH domain: new insights into the membrane docking reaction.. Biophys J, [PMID:19167305] |
10. Tessier PM, Lindquist S. () Prion recognition elements govern nucleation, strain specificity and species barriers.. Nature, [PMID:17495929] |