Do you have additional information on LIGHTNING LINK CONJUGATION KITS?

The reaction will be complete after a 3 hour incubation period at room temperature (20 - 25 degrees Celsius), 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.

Antibody concentration is critical for the Lightning-Link(TM) conjugation reaction, and has been optimized for each kit.  Therefore we suggest you consult the relevant instruction manual.

Yes.  The conjugation chemistry involves free amine groups, therefore any protein present in the mixture will become labeled during the Lightning-Link(TM) reaction.  Antibodies which are present in ascites fluid, serum or hybridoma culture media should be avoided.  However, purified antibodies are perfect for Lightning-Link(TM) and do not pose any problems.

The entire conjugation reaction is contained within one tube.  Thus the common losses, such as those on columns during dilutions and upon concentration, simply don't occur.  Antibody recovery is nearly 100%.

This is not necessary.  Lightning-Link(TM) conjugations are highly efficient and the conjugation kits are designed to yield a low level of free label at the end of the reaction; no separation steps are required.

Lightning-Link(TM) chemicals are designed to deactivate and are completely benign.  In Western Blots, ELISA, IHC, etc., you can use the conjugate right away.

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

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

Lightning Link(TM) 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.

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; these may deactivate the conjugation chemical which labels the protein of interest.  Common nucleophiles used in laboratories include amino acids (e.g. glycine), blockers (e.g. ethanolamine) and thiols (DTT, mercaptoethanol).

Concentrations of up to 0.5% BSA and 0.1% Gelatin have little effect on the conjugation chemistry.

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.

The AbSelect(TM) antibody purification kit enables you to remove such additives with ease.  Most importantly, the components of the AbSelect(TM) purification kit are fully compatible with the Lightning-Link(TM) conjugation system.  This means that the purified antibody can be added directly to any of the Lightning-Link(TM) kits.

Lightning-Link(TM) conjugates are just like any other conjugates.  For long-term storage at 4 degrees Celsius, you may wish to add antimicrobial agents or stabilizers (e.g. azide, BSA, glycerol, etc.)

Lightning-Link(TM) 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.

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.

Yes.  Conjugates formed with Lightning-Link(TM) are perfectly stable.

No.  Although Lightning-Link(TM) is a one-step conjugation method it has no similarity to other simple methods (e.g. glutaraldehyde) which tend to give ill-defined aggregates.

No.  Unlike conventional conjugation methods, Lightning-Link(TM) does not expose molecules to high pH.  The conjugations are carried out at physiological pH.