Antibody Conjugation

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Antibody Conjugation

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Antibody Conjugation

Methods for antibody modification have been developed in many previous studies with variety of labeling groups or conjugations. It is an important step for any further conjugation. In order to keep the functionality of the antibody after conjugation, modification is needed to avoid the conjugation to the immunoglobulin fractions.

 

Many molecules can be conjugated to antibody such as enzymes, nanoparticles, biotin, fluorescent molecules and so on. Fluorescein (FITC), a small organic molecule excited by the 488 nm and emission is 530 nm, is one of the most widely-used fluorescent dye for in vitro fluorescent imaging and FACS analysis. FITC is conjugated to proteins by primary amines in protein such as lysines. Previous studies show that about 5 FITC molecules (however, this is not an exactly number) are conjugated to one antibody molecule. So, the ratio of FITC/antibody should be optimized for best signal to noise ratio in detection and this protocol recommends 40-80 µg per mg of antibody.

Here is a protocol based on an original protocol devised by Aaron Kantor for FITC labeling. It will take about a half-day to perform.

Materials

 

NHS-FITC
DMSO
"Reaction Buffer":500 mM carbonate, pH 9.5
"Storage Buffer":10 mM Tris, 150 mM NaCl, 0.1% NaN3, pH 8.2

Preparation of antibody

  1. Remove any molecules that can react with FITC by dialysis or other method if necessary.
  2. Measure the antibody concentration and dilute the antibody to a concentration of 4 mg/ml.

Procedure

  1. Dissolve 10 mg of FITC in 1 mL anhydrous DMSO and make sure the solution is fresh prepared.
  2. Mix FITC with antibody immediately at a ratio of 40-80 µg/mg.
  3. Incubate and rotate at room temperature for at least 1 h protecting from light.
  4. Remove the free FITC by dialysis to separate the conjugates.
  5. Determine F/P ration and protein concentration by measuring the absorbance at 280 and 495 nm.

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