How to conjugate your antibody in one easy step
1. Which label should I use?
The choice of label for your antibody primarily depends upon the application:
- Enzymes (usually HRP or Alkaline Phosphatase)
- Fluorescent proteins and dyes
- Fluorescent proteins and dyes
- Fluorescent proteins
- Fluorescent dyes
- Tandem dyes
Please see the table here for absorption/emission wavelengths of Lightning-Link® fluorescent dyes, proteins and tandems.
2. What is the difference between Lightning-Link® and Lightning-Link® Rapid?
Both Lightning-Link® and Lightning-Link® Rapid kits can generate conjugates in a hands-on time of typically 20-30 seconds for the entire procedure. However the conjugation and quenching incubation times are shorter for Lightning-Link® Rapid kits allowing the conjugates to be generated faster. The antibody considerations are exactly the same, as is the high quality of the final conjugate.
3. What can I label using Lightning-Link®?
Lightning-Link® technology works by targeting free amine groups on your target. It can be used to label antibodies, peptides, proteins and any other molecules with free amine groups.
For advice on labeling molecules other than antibodies please contact our technical support team.
4. How pure does my antibody need to be?
Your antibody must be purified because other molecules with free amine groups will interfere with the reaction resulting in a poor quality conjugate.
A suitable method of purification should be selected:
- Affinity purification – this is the preferred method as it will always result in a purified sample containing only the antibody of interest.
- Protein A or G purification of serum – this will result in a purified sample of all IgG’s contained in the serum. You will have a purified IgG fraction but only a small percentage of this fraction will be the IgG of interest. This IgG fraction will label with our kits, although adjustments in conjugate dilutions will be required.
- Protein A or G purification of tissue culture supernatant or ascites fluid – this method will generate purified antibodies equivalent to affinity purification.
Other purification methods, such as ion exchange chromatography and ammonium sulphate precipitation, are low quality purification methods and should be avoided. Filtration using a 0.2/0.22 µm filter is a method of sterilization not purification, and is therefore not appropriate.
5. Is my antibody in a suitable buffer?
For recommended buffer conditions please see the below table:
1 Please note that individually the concentrations shown should not affect the reaction. However in combination with additional compounds that are not recommended above a certain concentration, the reaction may be affected.
2 If intending to use this kit for immunohistochemistry, it is recommended that there be no gelatin or BSA present.
3 It is important to note that sodium azide is a known inhibitor of HRP and should be avoided.
6. What can I do if my antibody formulation does not fit the requirements?
If your antibody requires purification or the buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link® kits.
The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume, etc.). We have designed a handy flow chart on the AbSelect™ webpage to help you select a kit, but feel free to contact us if you require further guidance. Please consult the individual kit protocols to see the antibody amount/volume suitable for each kit.
If your antibody is already purified but its concentration is significantly lower than 1mg/ml, you can concentrate it using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.
If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.
NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.
7. Do I need a wash or desalt step?
One of the advantages of the Lightning-Link® technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link® reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. This remaining free label would have its reactive ‘Lightning-Link®’ groups blocked by the Quencher provided in the kit, and is washed away during the wash step of your application.
8. How stable will my new conjugate be?
Upon dissolution of the Lightning-Link® mixture with a solution of the antibody, proprietary chemicals in the mixture become activated. This results in the directional covalent bonding of the antibody to the label in a gentle and controlled process at near-neutral pH. The stability of your conjugate is dependent on your antibody and label of choice.
Most Lightning-Link® conjugates can be stored for up to 18 months, undiluted at 4°C. The exception to this is tandem conjugates which are only stable for up to 3 months.