Antibody Labeling Guide (327-0015)

Lightning-Link Rapid Conjugation System
For product series 310-XXXX to 390-XXXX

Storage and components:
These kits are shipped at ambient temperature in a tamper-evident polypropylene container. Store at -20C upon receipt.

Kit contents:
Glass vial(s) of Lightning-Link Rapid mix (1 or 3 vials, depending on pack size)
1 vial of LL-Rapid Modifier reagent
1 vial of LL-Rapid Quencher reagent

Pre-conjugation considerations
Antibody buffer:
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 Lightning-Link Rapid 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.)

Recommended amount and volume of antibody for optimal results

Pack size Amount of antibody Volume of antibody
Up to 20ug 10-20ug 4-10ul
Up to 200ug 100-200ug 40-100ul
Up to 2mg 1-2mg 400-1000ul

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

Conjugation reaction
1. Before adding the antibody to the Lightning-Link Rapid mix, add 1ul of LL-Rapid Modifier reagent to each 10ul of antibody to be labeled. Mix gently.
2. Remove cap from vial of Lightning-Link Rapid mix and pipette the antibody sample (with added LL-Rapid Modifier) directly onto the lyophilised material. Resuspend gently by withdrawing and re-dispensing the liquid once or twice using a pipette.
3. Replace cap on the vial and leave standing for 15 minutes in the dark at room temperature (20-25C). Conjugations can also be set up and left overnight; longer incubation times have no negative effect on the conjugate.
4. After incubating for 15 minutes (or more), add 1ul of LL-Rapid Quencher reagent for every 10ul of antibody used and mix gently. The conjugate can be used after 4 minutes. The conjugates do not require purification.

Storage of conjugates
Storage at 4C 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.