trFluor™ Eu Acceptor XL665

1. Reduction of Antibody
2. Activate trFluor™ Eu Acceptor XL665
3. Reduced Antibody Conjugation with activated trFluor™ Eu Acceptor XL665
4. Purification

1. MTA Modification of Antibody
2. FOL Modification of trFluor™ Eu Acceptor XL665
3. MTA-Activated Antibody Conjugation with FOL-Activated trFluor™ Eu Acceptor XL665
4. Purification (optional with this Buccutite method)

1. Prepare a fresh 1.0 M DTT solution, by dissolving 15.4 mg of DTT in 100 µL of distilled water.

2. Prepare 0.7 mg of antibody, ensuring the concentration is at 1 mg/mL or higher for optimal results.

   Note: The reduction can be carried out in various buffers, such as MES, phosphate, or TRIS, with a pH range of 6 to 8.

3. Add 1.4 µL of 1.0 M DTT solution to 70 µL of the antibody solution and mix thoroughly. Allow the mixture to sit at room temperature for 30 minutes without further mixing to prevent cysteines from reoxidizing into cystines.

4. Purify the reduced antibody using a desalting column pre-equilibrated with 50 mM MES buffer (pH 6.0–6.5) supplemented with 2 mM EDTA.

5. Measure the antibody concentration using a Nanodrop spectrophotometer. Use the formula:

   Concentration (mg/ml) = A280nm / 1.4

   Note: Reduced antibodies are unstable. Perform the conjugation reaction immediately after purification to ensure optimal results.

1. Reconstitute trFluor™ Eu Acceptor XL665 in 100 µL ddH2O to make a 10 mg/mL.

   Note: The reconstituted trFluor™ Eu Acceptor XL665 can be stored at 4^oC for up to one month, protected from light.

2. Prepare a 10 mM stock solution of Sulfo-SMCC or SMCC in DMSO. Add 10–15 µL of the stock solution per 1 mg of trFluor™ Eu Acceptor XL665, mix thoroughly, and allow the reaction to proceed for 60 minutes.

3. Purify the activated trFluor™ Eu Acceptor XL665 using a desalting column that has been pre-equilibrated with 50 mM MES buffer (pH 6.0–6.5) containing 2 mM EDTA.

1. Mix the reduced antibody directly with the pre-activated trFluor™ Eu Acceptor XL665 at the ratio of 1 mg trFluor™ Eu Acceptor XL665/0.7mg antibody.

   Note: If necessary, adjust the antibody amount proportionally to maintain this ratio.

2. Allow the reaction to proceed for 60–120 minutes at room temperature.

1. The antibody/trFluor™ Eu Acceptor XL665 conjugate can be further purified using size exclusion chromatography to achieve optimal performance.

   Note: Store the antibody/trFluor™ Eu Acceptor XL665 conjugate solution at 2–8°C, protected from light.

    

    

    

Reagents needed but not provided:

Note: This protocol uses an antibody as the protein of choice for the conjugation example. If you are conjugating trFluor™ Eu Acceptor XL665 with a different protein, adjust the protein quantity according to its molecular weight.

1. Prepare a 10 mM stock solution of MTA, SE in DMSO.

2. Prepare 0.7 mg of antibody in PBS, ensuring the concentration is at 1 mg/mL or higher for optimal results.

3. Add 3.5 µL of 1.0 M NaHCO₃ buffer (pH 8.5) to 70 µL of the antibody solution and mix thoroughly. Then, add 4.5–7.5 µL of MTA, SE to the antibody solution, mix well, and incubate at room temperature for 60 minutes.

4. Purify the MTA-modified antibody using a desalting column that has been pre-equilibrated with 1X PBS.

1. Reconstitute trFluor™ Eu Acceptor XL665 in 100 µL ddH2O to make a 10 mg/mL.

   Note: The reconstituted trFluor™ Eu Acceptor XL665 can be stored at 4^oC for up to one month, protected from light.

2. Prepare a 10 mM FOL, SE stock solution in DMSO by adding 8–10 µL of a 10 mg/mL FOL, SE solution to 1 mg of trFluor™ Eu Acceptor XL665. Mix thoroughly and allow the reaction to proceed for 60 minutes.

3. Purify the FOL-activated trFluor™ Eu Acceptor XL665 using a desalting column pre-equilibrated with PBS buffer, such as the ReadiUse™ Bio-Gel P-6 spin column (Cat No. 60500).

1. Mix the MTA-activated antibody directly with the pre-activated trFluor™ Eu Acceptor XL665 at the ratio of 1 mg trFluor™ Eu Acceptor XL665/0.7mg antibody.

   Note: If necessary, adjust the antibody amount proportionally to maintain this ratio.

2. Allow the reaction to proceed for 60–120 minutes at room temperature.

1. The antibody/trFluor™ Eu Acceptor XL665 conjugate can be further purified using size exclusion chromatography to achieve optimal performance.

   Note: Store the antibody/trFluor™ Eu Acceptor XL665 conjugate solution at 2–8°C, protected from light.

Reagents needed but not provided: