Lightning-Link™ Frequently Asked Questions

METHODOLOGY

• Is the hands-on time really only 30 seconds?
Yes. Naturally you will need to familiarize yourself with the instructions before using Lightning-Link™ products for the first time, but after that your hands-on time should be no more than 30 seconds.


• How long does the entire conjugation reaction take?
The reaction will be complete after 3 hours at room temperature (20-25°C) with antibodies but a longer incubation time will not be detrimental. It is often extremely convenient to set up reactions overnight at room temperature and use the conjugate first thing the next morning.


• What is the ideal starting concentration of the antibody?
Antibody concentration is critical for the Lightning-Link™ conjugation reaction, and has been optimised for each kit. Therefore we suggest you consult the relevant instruction manual.


• Does the antibody have to be purified?
Yes – the conjugation chemistry involves free amine groups, therefore any protein present in the mixture will become labeled during the Lightning-Link™ reaction. Therefore antibodies which are present in ascites fluid, serum or hybridoma culture media should be avoided. However, purified antibodies are perfect for Lightning-Link™ and do not pose any problems.


• What recovery can I expect?
The entire conjugation reaction is contained within one tube. Thus the usual losses on columns, dilution of samples, and further losses upon concentration simply don't occur. Recoveries are therefore as close to 100% as you are ever likely to achieve.


• How do I separate out free label?
You don’t need to. Lightning-Link™ conjugations are highly efficient and the conjugation kits are designed to give a low level of free label at the end of the reaction. Thus no separation steps are required.


• Do I need to desalt the final conjugate?
Lightning-Link™ chemicals are designed to deactivate and are completely benign. In westerns, ELISA, IHC etc you can use the conjugate straight away.


• How scalable is the technology?
Scalability is one of the major advantages of Lightning-Link™. Rather than risk valuable antibody you can label a small quantity and be confident that the bulk quantity made later will have essentially identical properties. By avoiding desalting/dialysis steps the major source of losses and batch-to-batch variations in conjugation experiments are eliminated.


• Are other quantities available for scale up?

The standard packs address the need for a technology that allows conjugations to be performed on a microgram to milligram scale, but lyophilised material can be provided in any quantity with fast turnaround.

ANTIBODY BUFFERS

• What buffers can be used?
Lightning-Link™ works with phosphate, Hepes, MES and MOPS and other amine-free buffers. Conjugates can also be prepared in the presence of up to 20mM Tris buffer with almost no reduction in coupling efficiency. Once the reaction is complete the conjugate can be diluted in any buffer that is compatible with both the label and the antibody.


• What buffer additives can be used and what should be avoided?
Additives such as salts (e.g NaCl), sugars (e.g. sucrose) and chelators (e.g. EDTA) have no effect on the conjugation reaction. The main additives to avoid are nucleophiles that might deactivate the chemical, which conjugate the label to the protein of interest. Common nucleophiles used in laboratories include amino acids (e.g. glycine), blockers (e.g. ethanolamine) and thiols (DTT, mercaptoethanol).


• The antibody storage buffer contains common additives such as Bovine Serum Albumin (BSA) or gelatin. Will this affect conjugation chemistry?
Concentrations of up to 0.5% BSA and 0.1% Gelatin have little effect on the conjugation chemistry – please refer to the datasheets:

Lightning-Link™ conjugations in the presence of BSA datasheet
Lightning-Link™ conjugations in the presence of gelatin datasheet


• Does sodium azide cause interference?
A slight reduction in efficiency has been observed in head-to-head trials for samples with and without azide but for all practical purposes this is of little significance.
Lightning-Link™ conjugations in the presence of sodium azide datasheet


• How do I remove additives from the antibody storage buffer, which are at a higher concentration than that recommended for the Lightning-Link reaction?
The AbSelect™ antibody purification kit enables you to remove such additives with ease. Most importantly, the components of the AbSelect™ purification kit are fully compatible with the Lightning-Link™ conjugation system. This means that the purified antibody can be added directly to any of the Lightning-Link™ kits.


• Do I need to add preservatives?
Lightning-Link™ conjugates are just like any other conjugates. For long term storage at 4°C you may wish to add antimicrobial agents or stabilizers (e.g azide, BSA, glycerol, etc).

CONJUGATION CHEMISTRY

• What functional groups do I need on my protein to use Lightning-Link?
Lightning-Link™ requires amine groups on the molecule to be labeled. Most proteins or peptides have lysine and/or alpha-amino groups. All antibodies have multiple amine functions.


• Can proteins other than antibodies be labeled?
Yes. While labeling of primary antibodies is a major application, the only requirement is that the protein or molecule to be labeled has amine functionality.


• Is the label attached to my protein by a covalent bond?
Yes. Conjugates formed with Lightning-Link™ are perfectly stable.


• Will my antibody couple to itself or form high molecular weight aggregates?
No. Although Lightning-Link™ is a one-step conjugation method it has no similarity to other simple methods (e.g. glutaraldehyde) which tend to give ill-defined aggregates.


• Is my protein exposed to high pH?
No. Unlike conventional conjugation methods Lightning-Link™ does not expose molecules to high pH. Conjugations are carried out at physiological pH.


• How can I be kept updated on developments?

By signing up for the monthly e-mailer called first for bioconjugation. Please enter your email address into the box below.