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. 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.

[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.

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; ...; 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.)