| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Fluorescen dye; NHS-5(6)Carboxyrhodamine itself does not target specific biological receptors or enzymes. Instead, it functions as a fluorescent probe by covalently coupling with primary amino groups on biomolecules . Its NHS ester group reacts with ε-amino groups of lysine residues or N-terminal α-amino groups on proteins, peptides, or antibodies under physiological pH conditions to form stable amide bonds, thereby covalently attaching the rhodamine fluorophore to target molecules for detection and tracking . The compound may be non-fluorescent or weakly fluorescent in its unreacted state, with fluorescence enhancement upon reaction with target molecules, helping to reduce background signals .
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|---|---|
| ln Vitro |
Every NHS-5(6)Carboxyrhodamine/ConA sample shares the common property that the 556 nm peak is noticeably bigger than the 520 nm peak[1]. For NHS-5(6)Carboxyrhodamine, the dye/protein ratios obtained under natural conditions are similar to those produced under denaturing conditions[1].
In vitro, NHS-5(6)Carboxyrhodamine is used for fluorescent labeling of proteins and lectins. Studies have demonstrated that all NHS-5(6)Carboxyrhodamine/ConA (concanavalin A) samples exhibit a common characteristic: the 556 nm peak is significantly larger than the 520 nm peak . For NHS-5(6)Carboxyrhodamine, the dye-to-protein ratios obtained under native conditions are close to those obtained under denaturing conditions . The compound enables accurate dye-to-protein ratios under native conditions without the need for denaturation . |
| Enzyme Assay |
NHS-5(6)Carboxyrhodamine is used in cell-free systems for protein fluorescent labeling via bioconjugation techniques. A typical protocol includes: dissolving the target protein (e.g., ConA) in an appropriate labeling buffer (0.1 M sodium bicarbonate buffer, pH 8.5); dissolving NHS-5(6)Carboxyrhodamine in anhydrous DMSO to prepare a stock solution; adding the dye solution to the protein solution at an appropriate molar ratio (typically dye:protein = 5-20:1); incubating at room temperature in the dark for 1-2 hours; removing unreacted free dye by gel filtration chromatography or dialysis; detecting fluorescence signals of the labeled protein at 556 nm excitation to confirm labeling efficiency and dye-to-protein ratio .
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| ADME/Pharmacokinetics |
NHS-5(6)Carboxyrhodamine has a molecular weight of 527.53 g/mol as a small molecule fluorescent dye . For solubility, the compound is soluble in organic solvents such as DMSO but has limited water solubility . Labeling reactions are typically performed in aqueous buffers with low organic solvent content. Storage conditions: Powder is stable for 3 years at -20°C and 2 years at 4°C; solutions in solvent are stable for 1 year at -80°C and 1 month at -20°C, and should be stored protected from light to prevent photobleaching .
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| References | |
| Additional Infomation |
A new, relatively simple spectrophotometric method has been developed for accurately determining the extent of chromophore labeling in proteins. In dye–protein conjugates, the absorbance spectra and extinction coefficients are often significantly influenced by changes in the chromophore microenvironment, particularly at high dye/protein ratios. In the presented method, these microenvironmental effects are substantially reduced by denaturing the dye–protein complex in 6 M guanidine hydrochloride prior to spectrophotometric measurement.
Using this approach, extinction coefficients for tetramethylrhodamine isothiocyanate (TRITC) bound to a model protein receptor—the sugar-binding protein concanavalin A (ConA)—were determined under both native and denatured conditions. The extinction coefficients for TRITC/ConA conjugates were found to be 6.52 × 10⁴ M⁻¹ cm⁻¹ under native conditions and 6.96 × 10⁴ M⁻¹ cm⁻¹ under denaturing conditions. These values were obtained using a model dye complex formed between TRITC and ε-amino-n-caproic acid, which closely resembles the lysine side chain.
Additional dye–ConA conjugates were prepared using tetramethylrhodamine succinimidyl ester (RHS) and eosin isothiocyanate (EITC), and the effects of microenvironmental changes on these conjugates were also examined. For these dyes, the extinction coefficients measured under native and denaturing conditions showed no appreciable variation with the degree of labeling, indicating that changes in the microenvironment did not significantly affect their spectral properties.
In summary, this new method allows for straightforward and accurate determination of the dye/protein ratio for TRITC conjugates. Furthermore, RHS is expected to be a more suitable dye than TRITC for protein conjugation, as it enables more accurate determination of dye/protein ratios under native conditions. [1]
|
| Molecular Formula |
C29H25N3O7
|
|---|---|
| Molecular Weight |
527.5247
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| Exact Mass |
527.169
|
| CAS # |
150408-83-6
|
| Related CAS # |
5(6)-TAMRA SE;246256-50-8; 98181-63-6
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| PubChem CID |
12142572
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| Appearance |
Typically exists as solid at room temperature
|
| Density |
1.5±0.1 g/cm3
|
| Boiling Point |
759.4±70.0 °C at 760 mmHg
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| Flash Point |
413.1±35.7 °C
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| Vapour Pressure |
0.0±2.6 mmHg at 25°C
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| Index of Refraction |
1.710
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| LogP |
2.36
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
9
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
39
|
| Complexity |
982
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| Defined Atom Stereocenter Count |
0
|
| SMILES |
CN(C)C1=CC2=C(C=C1)C3(C4=C(C=C(C=C4)C(=O)ON5C(=O)CCC5=O)C(=O)O3)C6=C(C=C(C=C6)N(C)C)O2.CN(C)C1=CC2=C(C=C1)C3(C4=C(C=C(C=C4)N(C)C)O2)C5=C(C=CC(=C5)C(=O)ON6C(=O)CCC6=O)C(=O)O3
|
| InChi Key |
CXYYHBMOVJJZTD-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H25N3O7/c1-30(2)17-6-9-21-23(14-17)37-24-15-18(31(3)4)7-10-22(24)29(21)20-8-5-16(13-19(20)28(36)38-29)27(35)39-32-25(33)11-12-26(32)34/h5-10,13-15H,11-12H2,1-4H3
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| Chemical Name |
(2,5-dioxopyrrolidin-1-yl) 3',6'-bis(dimethylamino)-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylate
|
| Synonyms |
150408-83-6; NHS-5(6)Carboxyrhodamine; DTXSID90478456; RefChem:365504; DTXCID90429266;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8957 mL | 9.4783 mL | 18.9566 mL | |
| 5 mM | 0.3791 mL | 1.8957 mL | 3.7913 mL | |
| 10 mM | 0.1896 mL | 0.9478 mL | 1.8957 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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