Lightning-Link® Antibody Barcoding 5’ Series FAQ
1. What application(s) is Lightning-Link® Antibody Barcoding 5’ Series suited for?
Lightning-Link® Antibody Barcoding 5’ Series is suited for conjugation of antibodies of choice to barcoded oligonucleotides designed for Genomic Cytometry techniques using the format below:
- X represents the nucleotides of the barcode; each sequence is unique to Lightning-Link® Antibody Barcoding Kit – 5’ Series.
- N represents a randomly selected A, C, G, or T.
- The symbol * indicates a phosphorothioated bond, this modification renders the internucleotide linkage resistant to nuclease degradation.
2. What is the difference between Lightning-Link® Antibody Barcoding and Thunder-Link® PLUS?
Thunder-Link® PLUS is a universal oligo conjugation kit that enables simple and rapid conjugation of antibodies to any oligonucleotides of choice to suit any applications. The oligonucleotides need to be sourced separately. While Lightning-Link® Antibody Barcoding enables simple and rapid conjugation of antibodies to oligonucleotides in a specific format, usually dedicated to a specific application as described above. The oligonucleotides are included in the kits lyophilized. Lightning-Link® Antibody Barcoding requires even less hands-on time than Thunder-Link® PLUS and enables conjugation of as little as 10 µg per antibody with higher recovery.
3. Are all barcode sequences in Lightning-Link® Antibody Barcoding 5’ Series unique?
Yes, all of the barcode sequences in Lightning-Link® Antibody Barcoding 5’ Series are unique.
4. What does the pack sizes stand for?
10×10 μg – Barcodes A001-A010, B011-B020 and C021-C030 are 3 packs of 10 conjugation kits, each with 10 unique oligonucleotide sequences. These 3 packs can be used separately or together in any combination to build antibody panels with 10 μg conjugations. There are 30 unique oligonucleotide sequences available in total.
5×100 μg – Pick&Mix 5 Barcodes is a custom kit where any of the 30 unique oligonucleotide sequences available can be selected for 100 μg conjugations.
More information and sequences can be found in the Documents tab in the file Additional Sequences information Lightning-Link® Antibody Barcoding Kits 5′ Series.
5. 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, proteins, peptides, and any other molecules with free amine groups. However, the optional conjugate clean-up reagent to remove free oligos is only suitable for antibodies, so for proteins, peptides and other molecules, a bespoke conjugate clean-up would need to be developed if free oligos are likely to be an issue in your application.
For advice on labeling molecules other than antibodies please contact our technical support team.
6. 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.
7. Is my antibody in a suitable buffer?
For recommended buffer conditions please see the below table:
|Buffer Components & Conditions|
|Amine free buffer (e.g. MES, MOPS, HEPES, PBS)||Yes|
|Non-buffering salts (e.g. sodium chloride)||Yes|
|Chelating agents (e.g.EDTA)||Yes|
|Thiomersal / Thimerosal||No|
|Borate buffer||Not tested|
|Nucleophilic components (Primary amines e.g. amino acids or ethanolamine and thiols e.g. mercaptoethanol or DTT)||No|
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.
Compounds above marked ‘not tested’ yet are expected to be tolerated but no confirmatory data is available at this time. Please note that this table refers to expected impact in conjugation. You should also give due consideration to effects of these substances in your application, especially if the optional purification step is not required.
8. 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 to help you select a kit (view here) 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 1 mg/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.
9. Do I need to clean up my conjugate?
As there are many different applications only some general guidelines can be given. If free oligo is likely to be problematic, you will need to clean up the conjugate. The kit includes an optional conjugate clean-up reagent and protocol to remove free oligos.
10. What is the antibody:oligo conjugate yield after purification?
The antibody:oligo conjugate yield post-purification is dependent upon the operator’s accuracy and the number of purification steps. After two rounds, it was found that the yield is typically over 85%.
11. 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.
For any new antibody:oligo conjugate, initial storage at 4⁰C is recommended. A preservative may be desirable for long-term storage. Other storage conditions (e.g. frozen at -70⁰C or at -20⁰C with 50% glycerol) may also be satisfactory. The best conditions for any new conjugate must be determined by experimentation. You should also take account of how the conjugate will be used and avoid adding substances that will only need to be removed later, resulting in inevitable losses of conjugate.
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