Fluorescent dye; 5(6)-TAMRA itself does not target specific biological receptors or enzymes. Instead, it functions as a fluorescent probe by covalently coupling with biomolecules. Its carboxylic acid group can be activated by carbodiimide (e.g., EDC) to react with primary amines on biomolecules (proteins, peptides, nucleic acids), forming stable amide bonds that enable fluorescent labeling and tracking of target molecules. TAMRA's fluorescence properties arise from its rhodamine core structure, which absorbs light at a specific wavelength and re-emits it at a longer wavelength—a process called fluorescence.

Stock Solution Preparation
1. Protein Preparation
To achieve optimal labeling efficiency, adjust the protein (antibody) concentration to 2 mg/mL.
1) The pH of the protein solution should be 8.5 ± 0.5. If the pH is below 8.0, adjust it using 1 M sodium bicarbonate.
2) If the protein concentration is below 2 mg/mL, labeling efficiency will be significantly reduced. A final protein concentration in the range of 2–10 mg/mL is recommended for optimal labeling efficiency.
3) The protein must be in a buffer that does not contain primary amines (e.g., Tris or glycine) or ammonium ions, as these may interfere with labeling efficiency.

2. Dye Preparation
Dissolve the 5(6)-TAMRA dye in anhydrous DMSO to prepare a 10 mM stock solution. Mix thoroughly using a glass tube or by vortexing.
Note: It is recommended to aliquot the 5(6)-TAMRA stock solution and store at –20 °C or –80 °C protected from light. Prior to use, the dye must be activated with a conjugation buffer (500 μg/mL) before proceeding with the labeling reaction.

3. Calculation of Dye Working Solution Volume
The amount of 5(6)-TAMRA dye required for the labeling reaction depends on the amount of protein to be labeled. The optimal molar ratio of 5(6)-TAMRA dye to protein is approximately 10.
Example: To label 500 μL of IgG at 2 mg/mL (MW = 150,000), dissolve 1 mg of 5(6)-TAMRA dye in 100 μL DMSO. The required volume of 5(6)-TAMRA is 2.88 μL. The calculation procedure is as follows:
1) mmol (IgG) = mg/mL (IgG) × mL (IgG) / MW (IgG) = 2 mg/mL × 0.5 mL / 150,000 mg/mmol = 6.7 × 10⁻⁶ mmol
2) mmol (5(6)-TAMRA) = mmol (IgG) × 10 = 6.7 × 10⁻⁶ mmol × 10 = 6.7 × 10⁻⁵ mmol
3) μL (5(6)-TAMRA) = mmol (5(6)-TAMRA) × MW (5(6)-TAMRA) / mg/μL (5(6)-TAMRA) = 6.7 × 10⁻⁵ mmol × 430.45 mg/mmol / 0.01 mg/μL = 2.88 μL

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Protocol
1. Labeling Reaction
1) Slowly add the calculated volume of freshly prepared 10 mg/mL 5(6)-TAMRA dye to 0.5 mL of the protein sample solution. Mix gently and briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid vigorous mixing to prevent protein denaturation and loss of activity.
2) Place the reaction tube in a dark place and incubate with gentle shaking at room temperature for 60 minutes. Every 10–15 minutes, gently invert the tube several times to thoroughly mix the reactants and improve labeling efficiency.

2. Protein Purification and Desalting
The following protocol uses a Sephadex G-25 column for purifying the dye–protein conjugate.
1) Prepare the Sephadex G-25 column according to the manufacturer’s instructions.
2) Load the reaction mixture onto the top of the Sephadex G-25 column.
3) When the sample has run just below the surface of the top resin, immediately add PBS (pH 7.2–7.4).
4) Add more PBS (pH 7.2–7.4) to elute the desired sample. Collect the fractions containing the desired dye–protein conjugate.

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In vitro, 5(6)-TAMRA is widely used for fluorescent labeling of biomolecules. In bacterial labeling experiments, 5(6)-TAMRA is used to label Salmonella typhimurium, allowing visualization and counting of adherent bacteria under fluorescence microscopy using a Cy3 filter. In protein-peptide interaction studies, TAMRA-labeled peptides (concentrations ranging from 10 nM to 5 μM) are used to detect binding affinities on SH2 and PTB domain protein microarrays. TAMRA exhibits fluorescence properties with excitation at approximately 541-560 nm, emission at approximately 565-583 nm, and an extinction coefficient >90,000 cm⁻¹M⁻¹.

5(6)-TAMRA is used to study molecular distribution and targeting in vivo. In neuroscience research, TAMRA-labeled orexin-A is used to observe co-internalization of the orexin-1 receptor by monitoring the binding and trafficking of the TAMRA-labeled ligand. In tumor imaging studies, TAMRA fluorescent labeling coupled with IL-13 peptide-conjugated Gd₃N@C₈₀ nanoparticles (TAMRA-IL-13-f-Gd₃N@C₈₀) is used for MRI studies of IL-13Rα2 expression on glioma tumors in mice.

5(6)-TAMRA is primarily used in cell-free systems through bioconjugation techniques. A typical protein labeling protocol involves: dialyzing the target protein (e.g., collagen I) overnight against 0.25 M sodium bicarbonate labeling buffer (containing 0.4 M NaCl, pH 9.5); mixing 10 mg/mL TAMRA solution with the dialyzed protein and incubating overnight with rotation; removing excess free dye by dialysis; storing the labeled product at 4°C protected from light. In protein microarray assays, TAMRA-labeled peptides (10 nM-5 μM) are incubated with immobilized protein domains in buffer containing 20 mM Hepes, 100 mM KCl, 0.1% Tween-20 (pH 7.8) for 1 hour, and binding signals are detected by fluorescence scanning.

5(6)-TAMRA is widely used for fluorescence labeling and tracking at the cellular level. In Salmonella infection experiments, bacterial cells labeled with 5(6)-TAMRA are used to infect MDCK-1 cell monolayers or microtissues for 3 hours at 37°C with 5% CO₂. After infection, samples are fixed with 3.7% formaldehyde, and images are acquired using a Leica fluorescence microscope with a Cy3 filter and 40x objective. Adherent bacteria are manually counted per field of view. In microglial phagocytosis assays, UV-stressed hippocampal neurons are stained with 5(6)-TAMRA SE and then co-cultured with adult mouse microglia for 2 hours at 37°C. After fixation with 2.5% PFA, phagocytosis efficiency is analyzed by Iba1 immunofluorescence staining and confocal microscopy.

5(6)-TAMRA and its derivatives are used for molecular tracing in animal models. In protein-peptide interaction studies, TAMRA-labeled peptides are used for binding analysis on SH2 and PTB domain microarrays. Peptides at various concentrations (5 μM, 3 μM, 2 μM, 1 μM, 500 nM, 200 nM, 100 nM, and 10 nM) are incubated for 1 hour, and fluorescence signals are scanned to determine binding affinities. In probiotic colonization studies, TAMRA-labeled probiotics are administered orally to mice, and intestinal survival and colonization are assessed by monitoring fecal fluorescence intensity.

5(6)-TAMRA has a molecular weight of 430.45 g/mol as a small molecule fluorescent dye. Its fluorescence spectral properties are Ex/Em ~541-560/565-583 nm, with stability in buffers at pH 7-9. For solubility, it is soluble in DMSO at ≥23.83 mg/mL, soluble in ethanol at ≥4.45 mg/mL (requires gentle warming and ultrasonication), and insoluble in water. As a small molecule dye, TAMRA may diffuse freely in aqueous environments and may be rapidly excreted or metabolized in vivo. When conjugated to larger molecules like proteins or nucleic acids, its ADME properties are more influenced by the properties of the conjugated molecule. Storage conditions: Powder is stable for 3 years at -20°C; solutions are stable for 6 months at -20°C and should be stored protected from light to prevent photobleaching.

According to GHS classification, 5(6)-TAMRA is not classified as a hazardous substance or mixture. This product is for research use only and is not intended for diagnostic or medical purposes. In cell and animal studies reported in the literature, TAMRA-labeled molecules did not exhibit明显的 toxic effects under the experimental conditions, although the specific toxicity profile depends on the conjugated molecule and the experimental system.

[1]. Pathway for polyarginine entry into mammalian cells. Biochemistry. 2004 Mar 9;43(9):2438-44.

[2]. Insertion and organization within membranes of the delta-endotoxin pore-forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide. J Biol Chem. 2000 Aug 4;275(31):23602-7.

[3]. Automated, solid-phase coupling of rhodamine dye acids to 5' amino oligonucleotides. Nucleic Acids Symp Ser. 2000;(44):257-8.

5-carboxytetramethylrhodamine is a tetramethylrhodamine compound having a carboxy substituent at the 5-position. It has a role as a fluorochrome.

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5(6)-TAMRA (CAS: 98181-63-6) and 5(6)-TAMRA SE (CAS: 246256-50-8) are different products. TAMRA contains a carboxylic acid group and requires EDC activation before reacting with primary amines; in contrast, TAMRA SE contains a succinimidyl ester group and can react directly with primary amines without the need for an additional activation step. TAMRA is a commonly used fluorescent labeling group in the synthesis of PROTAC linkers and can be employed for fluorescent labeling of PROTAC molecules and cellular uptake studies. This dye is sensitive to light and temperature and must be stored under strict protection from light and at low temperatures to preserve its fluorescence properties.

C25H22N2O5

430.45

430.15287

98181-63-6

5(6)-TAMRA SE;246256-50-8

2762602

Brown to black solid powder

≥300ºC(lit.)

9.059

1

6

3

32

875

0

CN(C)C1=CC2=C(C=C1)C(=C3C=CC(=[N+](C)C)C=C3O2)C4=C(C=C(C=C4)C(=O)[O-])C(=O)O

YMZMTOFQCVHHFB-UHFFFAOYSA-N

InChI=1S/C25H22N2O5/c1-26(2)15-6-9-18-21(12-15)32-22-13-16(27(3)4)7-10-19(22)23(18)17-8-5-14(24(28)29)11-20(17)25(30)31/h5-13H,1-4H3,(H-,28,29,30,31)

3-carboxy-4-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]benzoate

5-carboxytetramethylrhodamine; CHEBI:51657; RefChem:537820; 91809-66-4; TAMRA Acid; 5(6)-TAMRA Acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 20.83 mg/mL (~48.39 mM)

Solubility in Formulation 1: 2.08 mg/mL (4.83 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)