Labeling non-antibody proteins and small molecules – An Advanced Guide

Lightning-Link® conjugation with non-antibody proteins and small molecules

The Lightning-Link conjugation system is the world’s easiest way of making conjugates with any one of around 50 labels. Lightning-Link was originally optimized for antibodies, but it has a much broader range of application. The following guide may help if you wish to label molecules other than antibodies.

Key considerations for non-antibody proteins

Lysine residues

The most important consideration is whether the protein has lysine residues. All antibodies have multiple lysine residues, but the same cannot be assumed for non-antibody proteins. Any concern over the number of lysines increases as the size of the protein decreases. However, in many/most cases the deduced amino acid sequence of the protein will be known, and the sequence can be inspected for lysine residues (K or Lys, depending on whether single-letter or three-letter code is used). For non-antibody proteins the efficiency of reaction cannot be predicted, and some initial optimization may be needed.

Size of protein and amount of protein to add to Lightning-Link vials

Lightning-Link protocols recommend the amount of antibody to add, as this is the most common use of the kit. However, the amount can be adjusted to suit other proteins. The molar ratio of label to protein is important, as excessive amounts of label may damage the protein or cause high backgrounds in the final assay. For example, if the protein to be labeled is 75 kDa (half the size of an antibody) the addition of 10ug of protein would introduce twice the amount in molar terms as with 10ug of antibody.  A consequence of this may be the presence of unlabeled protein at the end of the reaction. Whether this is a concern will depend on the assay application, but the addition of less protein (5ug in this case) would normally be a safer starting position.

Small proteins (but still >10 kDa)

In view of the approach recommended above for a 75kDa protein, a further reduction in the mass added to the vial may seem logical if the size of the protein becomes even smaller. However, the protein could be significantly smaller than some labels e.g. HRP 40 kDa, Phycoerythrin (240 kDa), alkaline phosphatase (160 kDa). Thus, one might consider that the label (L) is being labeled with the protein (P), rather than the protein with the label. Large labels may have several reactive surface groups and the label may be able to conjugate to several molecules of a small protein (i.e. conjugate structure L1Pn, where n = >1). In certain circumstances this may be advantageous, thus one would not automatically reduce the mass of protein simply because the protein is very small. In other cases, L1P1 may be required, in which case a high molar excess of P should not be used. An advantage of Lightning-Link is that conjugation reactions can be done at small scale (e.g. 10ug with antibodies), which is perfect for optimizing the conditions with non-antibody proteins.

Peptides/small molecules (<10Kda and often <1 kDa)  

With these molecules it is inevitable there will be few, if any, lysines. However, many peptides will have a free N-terminus i.e. with a primary amine on the first amino acid, which can participate in Lightning-Link reactions. In some cases, however, the N-terminus will be ‘blocked’ e.g. by acetyl groups, which may have been added during peptide synthesis to improve peptide stability in vivo (e.g. to prevent attack by aminopeptidases). A peptide that is both N-terminally blocked and lacks lysine residues cannot be conjugated using Lightning-Link technology.

Small molecules other than peptides may be compatible with Lightning-Link if they contain primary amines. These are -NH2 groups, but note that in some functional groups e.g. amide, -CO(NH2), the -NH2 element is strongly affected by the adjacent groups (carbonyl in this case) and the amide group is unreactive. Generally, if the -NH2 group is attached to a -CH2 group it is likely to be reactive. Indeed, in the case of lysine residues in proteins and antibodies, the lysine side chain has four -CH2 units followed by a terminal -NH2.

With either small molecules or small peptides, the size of the molecule compared with an antibody is not particularly relevant in determining what mass should be added to a Lightning-Link vial. Any protein labels e.g. HRP will be almost certainly conjugated with several small molecules; often this situation will be desirable, but it is necessary to consider the final application before deciding on how much small molecule should be added. If multiple small molecules need to be attached, you can add the molecule to 1mM concentration. This will result in efficient conjugation, but you may need to desalt the resulting conjugate i.e. if free small molecules are expected to interfere in the final assay. Alternatively, you can try to reduce the concentration of small molecule to a level where purification is not needed. If it is not possible to predict what sort of conditions will work best, a range of different concentrations/amounts can be explored.

Finally, the examples above cover many non-antibody scenarios but if your molecule to be labeled has any unusual features or you are not sure of how to proceed, please contact us here for further advice.