The Lightning-Link conjugation kit allows R-PC conjugations to set up in seconds, simply by adding a solution of the antibody to be labeled to a proprietary lyophilised mixture containing R-PC (Figure 1). By circumventing the desalting or dialysis steps that commonly interrupt traditional protein conjugation procedures, Lightning-Link technology can be used to label small quantities of protein for FACS analysis with 100% recovery.
Upon dissolution of Lightning-Link mixture with a solution of the antibody (or other biomolecule to be labeled) proprietary chemicals in the mixture become activated. This results in the directional, covalent bonding of the antibody to the protein label in a gentle and controlled process at near-neutral pH. The hands-on time for the entire procedure is usually 20-30 seconds. Lightning-Link makes it possible to label antibodies with R-PC with ease, and eliminates the need for secondary reagents in FACS experiments. Direct labeling can simplify and improve data quality in multicolor experiments by eliminating problems caused by dissociation and crossover of secondary reagents.
- Considerations before use
- Sample buffer
Ideally, the antibody to be labeled should be in 10-50mM amine-free buffer pH range 6.5 to 8.5. However, many buffers outside these limits of concentration and pH can be accommodated. Modest concentrations of Tris buffer are also tolerated. Appendix 1 gives further guidance on buffers and compatible additives.
- Amount and volume of antibody
The amount of antibody used for labeling should be the same as the pack size of LL-R-PC. e.g. For 100ug LL-R-PC add 100ug of antibody. The volume in which the antibody is added ideally should be 40ul-100ul (100ug pack size), and 0.40-1ml (1mg pack size). Antibody concentrations in the range 1-4mg/ml are therefore ideal. However, concentrations and volumes outside these suggested limits have also yielded excellent conjugates. For any new antibody, optimization of the ratio of antibody to R-PC is often worthwhile.
- Setting up conjugation reactions
- Before you add antibody to the Lightning-Link mix, add 1ul of LL-Modifier reagent for each 10ul of antibody to be labeled. Mix gently.
- Remove the screw cap from the vial of Lightning-Link mix and pipette the antibody sample (with added LL-modifier) directly onto the lyophilised material. Resuspend gently by withdrawing and re-dispensing the liquid once or twice using a pipette.
- Place the cap back on the vial and leave it standing for 3 hours in the dark at room temperature (20-25 degrees Celsius). Alternatively, and often more conveniently, conjugations can be set up and left overnight, as the longer incubation time has no negative effect on the conjugate.
- After incubating for 3 hours (or more), add 1ul of LL-quencher reagent for every 10ul of antibody used. The conjugate can be used after 30 minutes.
- Storage of conjugates
For any new conjugate, storage at 4 degrees Celsius is recommended. 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.
Appendix 1. Compatibility of buffers and buffer additives.
Amine-free buffers, including MES, MOPS, HEPES and phosphate are compatible if they are in the concentration range 10-50mM and have pH values in the range 6.5-8.5, as the addition of LL-Modifier provides the conditions necessary for efficient conjugation. Common non- buffering salts (e.g. sodium chloride), chelating agents (e.g. EDTA), and sugars have no effect on conjugation efficiency. Azide (0.02-.1%) has little or no effect. Glycerol up to 50% has no effect. If the amine-free buffer is relatively concentrated and outside the pH range 6.6-8.5 you may need to add more LL-modifier for each 10ul of antibody. Excess LL-Modifier is provided so that you can check the pH of the buffer after the addition of the modifier. Ideally the pH should be around 7.3-7.6, though efficient conjugation occurs anywhere between pH 6.8 and 7.8. Avoid buffer components that are nucleophilic, as these may react with Lightning-Link chemicals. Primary amines (e.g. amino acids, ethanolamine) and thiols (e.g. mercaptoethanol, DTT) fall within this class. (Note: Tris has little effect on conjugation efficiency as long as the concentration is 20mM or less).