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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.
The most important consideration when labeling your protein is whether it contains lysine residues, that are used to link the label.
All antibodies have multiple lysine residues, but the same cannot be assumed for non-antibody proteins. In most cases the 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 a specific amount of antibody to use, but this can easily be adapted to suit other proteins.
The molar ratio of label to protein is important, as excessive amounts of the 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 10 μg of protein would introduce twice the amount in molar terms as with 10 ug 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 (5 μg in this case) would normally be a safer starting position.
Small proteins (but still >10 kDa)
While we recommend scaling your protein volume for use in Lightning-Link reactions, some proteins are significantly smaller than the labels we want to use e.g. HRP (40 kDa), Phycoerythrin (240 kDa), alkaline phosphatase (160 kDa) and so require further consideration.
Large labels may have several reactive surface groups and the label may be able to conjugate to several molecules of a small protein.
An advantage of Lightning-Link is that conjugation reactions can be done at a small scale (e.g. 10 μg with antibodies), which is perfect for optimizing the conditions with non-antibody proteins.?
Peptides/small molecules (<10 kDa and often <1 kDa)
??With these molecules it is inevitable there may be very few, if any, lysine residues. However, most 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, 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.
When working with small molecules or small peptides, the size of the molecule compared with an antibody is not that relevant in determining what mass should be added to a Lightning-Link vial. Any protein labels e.g. HRP will be almost definitely be conjugated to several small molecules; often this situation will be beneficial, but it is importnat to consider the final application before deciding on how much small molecule you do add. If multiple small molecules need to be attached, you can add the molecule to 1 mM concentration. This will give 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 isn't possible to predict what sort of conditions will work best, you could explore a range of different concentrations/amounts to optimize your experiments.
The examples above cover the most common non-antibody uses of Lightning-Link, but if your molecule to be labeled has any unusual features or you are just not sure how to proceed, contact us here for further advice.