Fluorescent dye

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. For optimal labeling efficiency, a final protein concentration in the range of 2–10 mg/mL is recommended.
3) The protein must be in a buffer that does not contain primary amines (such as Tris or glycine) or ammonium ions, as these can interfere with labeling efficiency.

2. Dye Preparation
Dissolve the 5(6)-TAMRA SE dye in anhydrous DMSO to prepare a 10 mM stock solution. Mix thoroughly using a glass tube or by vortexing.

3. Calculation of Dye Working Solution Volume
The amount of 5(6)-TAMRA SE dye required for the labeling reaction depends on the amount of protein to be labeled. The optimal molar ratio of 5(6)-TAMRA SE dye to protein is approximately 10.
Example: To label 500 μL of IgG at 2 mg/mL (MW = 150,000), dissolve one vial of 1 mg 5(6)-TAMRA SE dye in 100 μL DMSO. The required volume of 5(6)-TAMRA SE is 3.53 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 SE) = mmol (IgG) × 10 = 6.7 × 10⁻⁶ mmol × 10 = 6.7 × 10⁻⁵ mmol
3) μL (5(6)-TAMRA SE) = mmol (5(6)-TAMRA SE) × MW (5(6)-TAMRA SE) / mg/μL (5(6)-TAMRA SE) = 6.7 × 10⁻⁵ mmol × 527.53 mg/mmol / 0.01 mg/μL = 3.53 μL

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Protocol
1. Labeling Reaction
1) Slowly add the calculated volume of freshly prepared 10 mg/mL 5(6)-TAMRA SE 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.

Bacterial labeling for concentration monitoring: Two bacterial strains isolated from the aquifer were cultured to the mid-logarithmic phase, washed twice with phosphate-buffered saline (PBS) to remove culture medium components. 5(6)-TAMRA SE was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, then diluted with PBS to a final concentration of 10 μM. Bacterial suspensions (1×10⁶ CFU/mL) were mixed with the dye solution and incubated at 25°C in the dark for 30 minutes. Unbound dye was removed by three rounds of centrifugation and resuspension in PBS. Labeled bacteria were detected using a fluorescence microscope and flow cytometer, with excitation at 544 nm and emission at 572 nm, enabling near-real-time concentration monitoring of the two strains simultaneously [1]
Peptide labeling for polymer incorporation: Synthetic peptides with free amino groups were dissolved in 0.1 M sodium bicarbonate buffer (pH 8.5). 5(6)-TAMRA SE (molar ratio of dye to peptide = 3:1) was added as a DMSO stock solution, and the mixture was stirred at room temperature in the dark for 2 hours. The reaction was quenched by adding 1 M glycine solution (10% of the reaction volume) and incubating for 30 minutes. Labeled peptides were purified by gel filtration chromatography followed by reverse-phase HPLC to remove unreacted dye and by-products. The labeling efficiency was confirmed by UV-Vis spectroscopy and mass spectrometry, with the labeled peptides showing characteristic fluorescence at 572 nm upon excitation [2]

[1]. Application of a vital fluorescent staining method for simultaneous, near-real-time concentration monitoring of two bacterial strains in an Atlantic coastal plain aquifer in Oyster, Virginia. Appl Environ Microbiol. 2004 Mar;70(3):1680-7.

[2]. Labelling peptides with fluorescent probes for incorporation into degradable polymers. Eur J Pharm Biopharm. 1998 May;45(3):265-73.

[3]. Design and synthesis of new acid cleavable linkers for DNA sequencing by synthesis. Nucleosides Nucleotides Nucleic Acids. 2014;33(12):774-85.

5(6)-TAMRA SE (5(6)-carboxytetramethylrhodamine succinimide ester) is a fluorescent dye derivative that exists as a mixture of 5- and 6-isomers [1][2]. It contains a succinimide ester (SE) functional group, which can covalently react with the primary amino group (-NH₂) of biomolecules (such as bacterial surface proteins and peptide amino termini) to form a stable amide bond, thereby achieving fluorescent labeling [1][2]. Its main fluorescent properties include an excitation wavelength of approximately 544 nm and an emission wavelength of approximately 572 nm, with high fluorescence quantum yield and photostability, making it suitable for long-term monitoring and detection [1][2]. Due to its strong fluorescence and stable coupling effect, typical applications include microbial labeling in environmental monitoring, peptide/protein labeling for polymer incorporation, and biomolecule tracing [1][2].

C29H25N3O7

527.5247

527.169

246256-50-8

5(6)-TAMRA;98181-63-6;NHS-5(6)Carboxyrhodamine;150408-83-6

92044448

Purple to purplish red solid powder

3.545

0

18

10

78

1960

0

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(O2)C=C(C=C6)N(C)C.CN(C)C1=CC2=C(C=C1)C3(C4=C(O2)C=C(C=C4)N(C)C)C5=C(C=CC(=C5)C(=O)ON6C(=O)CCC6=O)C(=O)O3

UWQYEQKHQZYMMZ-UHFFFAOYSA-N

InChI=1S/2C29H25N3O7/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;1-30(2)17-6-9-20-23(14-17)37-24-15-18(31(3)4)7-10-21(24)29(20)22-13-16(5-8-19(22)28(36)38-29)27(35)39-32-25(33)11-12-26(32)34/h2*5-10,13-15H,11-12H2,1-4H3

(2,5-dioxopyrrolidin-1-yl) 3',6'-bis(dimethylamino)-1-oxospiro[2-benzofuran-3,9'-xanthene]-5-carboxylate;(2,5-dioxopyrrolidin-1-yl) 3',6'-bis(dimethylamino)-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylate

246256-50-8; Xanthylium, 9-[2-carboxy-4(or 5)-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]phenyl]-3,6-bis(dimethylamino)-, inner salt; 2,5-dioxopyrrolidin-1-yl 3',6'-bis(dimethylamino)-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-5-carboxylate; 2,5-dioxopyrrolidin-1-yl 3',6'-bis(dimethylamino)-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-6-carboxylate; MFCD30179714; 5(6)-TAMRASE;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Nitrogen protection, the product is moisture-sensitive, minimize exposure to air, and avoid repeatedly taking it in and out of the refrigerator to prevent the impact of moisture condensation.

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

DMSO : ~10 mg/mL (~18.96 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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