xtraFluor™ Violet 420 Labeling Dye

1. Prepare a 1 mg antibody solution (preferably at a concentration of at least 2 mg/mL) by mixing 5% (v/v) of reaction buffer (such as 1 M sodium bicarbonate solution or 1 M phosphate buffer, pH ~8.5).
   

   Note: The pH of the antibody solution should be around 8.0-8.5. If the pH of the antibody solution is lower than 8.0, adjust it to bring within the range using either 1 M sodium bicarbonate solution or 1 M phosphate buffer at pH 8.5.

   Note: The antibody should be dissolved in 1X phosphate-buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, dialyze it against 1X PBS, pH 7.2-7.4, to remove any free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) commonly used in protein precipitation.

   Note: Antibodies that are impure or stabilized with bovine serum albumin (BSA) or gelatin may not label effectively. Additionally, sodium azide or thimerosal can interfere with the conjugation reaction. To achieve optimal labeling results, these preservatives should be removed through dialysis or spin column techniques.

   Note: For optimal labeling efficiency, it is recommended to maintain a final protein concentration between 2-10 mg/mL. Protein concentrations below 2 mg/mL can significantly reduce conjugation efficiency.

1. Add 20 µL of anhydrous DMSO directly to the vial of Buccutite™ MTA (100 µg/vial) to prepare 5 mg/ml stock solution. Mix well by pipetting or vortexing.

   Note: Prepare the Buccutite™ MTA stock solution before starting the conjugation, and use it promptly. Extended storage of the dye stock solution may reduce the dye activity. It can be stored in the freezer for up to two weeks, provided it is protected from light and moisture. Avoid freeze-thaw cycles.

1. Add 2.5 µL-3.5 µL of 5 mg/ml Buccutite™ MTA to 1mg of antibody and mix well by repeatedly pipetting for a few times or vortex the vial for few seconds. 
2. Incubate the Antibody- MTA reaction mixture at room temperature for 30 - 60 minutes.

3. Purify Antibody- MTA reaction mixture through desalting column.

4. Purify the conjugate on a gel filtration column, such as a Sephadex G-25 column or equivalent matrix, or by extensive dialysis at 4°C in PBS buffer.

5. Calculate the Antibody- Buccutite™ MTA concentration by estimating 80% yield after desalting. For example, if starting with 1 mg protein, after desalting column purification, the recovery protein amount is ~ 0.8 mg.

Recommended AAT Bioquest Desalting Columns:

Combine elutions if using multiple columns.

1. Conjugation reaction is initiated by mixing Antibody- MTA and xtraFluor™ Violet 420 at the ratio of 1mg antibody to 1mg xtraFluor™ Violet 420.
   
   Note: Add PBS to make sure the Ab concentration is 1.5- 2mg/ml in the reaction mixture.
   
   Note: The mix sequence is: mix dye and PBS first, and then add Antibody-MTA to the dye.
2. Incubate the mixture for 1~2 hour at room temperature or overnight at 4°C. The reaction mixture is now ready to use.
3. If required, the reaction mixture can be purified by size exclusion chromatography (SEC), and the desired conjugate fractions are pooled and combined.

1. Use Nanodrop to measure the absorption spectrum of the SEC purified Antibody–xtraFluor™ Violet 420 conjugate.
2. Read OD (absorbance) at 280 nm and dye maximum absorption (ƛ max = 407 nm). Calculate DOL and antibody concentration with the following parameters:
   Ec (407 nm) = 3,000,000 M^-1cm^-1
   CF280 = 0.067
   Ec (IgG) = 210,000 M^-1cm^-1