FITC Conjugation Kit (ab102884)

The antibody to be labelled should be purified, in an appropriate buffer for conjugation and at a suitable concentration, as described in the protocol booklet. If not, consider using our antibody purification and concentration kits.

Pre-conjugation considerations

The purified antibody to be labeled should ideally be in 10-50mM amine-free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5. If the buffer is more concentrated or outside this pH range contact the technical support team.

Common non-buffering salts (e.g. sodium chloride), chelating agents (e.g. EDTA), and sugars have no effect on conjugation efficiency. Azide (0.02 to 0.1%) and BSA (0.1 to 0.5%) have little or no effect. Glycerol up to 50% has no effect.

Avoid buffer components that are nucleophilic, as these may react with the kit chemicals. Primary amines (e.g. amino acids or ethanolamine) and thiols (e.g. mercaptoethanol or DTT) fall within this class (Note: Tris-based buffers should be avoided). If your buffer contains primary amines and/or thiols, you should consider using our Concentration and Purification Kits (ab102778 or ab102784).

Recommended amount and volume of antibody for optimal results

Antibody concentrations of 1-4 mg/ml generally give optimal results. For conditions outside the suggested ranges contact the technical support team.

Conjugation reaction

• Before adding the antibody to the FITC mix, add 1 µl of Modifier reagent to each 10 µl of antibody to be labelled. Mix gently.
• Remove cap from vial of FITC mix and pipette the antibody sample (with added  Modifier) directly onto the lyophilized material. Resuspend gently by withdrawing and re-dispensing the liquid once or twice using a pipette.

• Replace cap on the vial and leave standing in the dark at room temperature (20-25°C) for 3 hours. Conjugations can also be set up and left overnight; longer incubation times have no negative effect on the conjugate.

• After incubating for 3 hours (or more), add 1 µl of  Quencher  reagent for every 10 µl of antibody used and mix gently. The conjugate can be used after 30 minutes. The conjugates do not require purification.

Storage of conjugates

Storage at 4°C is recommended for any conjugate. A preservative may be desirable for long-term storage. Other storage conditions may also be satisfactory. The best conditions for any particular conjugate must be determined by experimentation.

Disclaimer

Labeling of the antibody will not work if the conjugation blocks the active paratope. This situation is rare.