Lightning-Link® R-PE Antibody Labeling Kit

Lightning-Link® R-PE Antibody Labeling Kit Summary

Applications/Dilutions

Packaging, Storage & Formulations

Kit Components

Notes

Background

Limitations

Publications for Lightning-Link (R) R-PE Kit (703-0010)(60)

Reviews for Lightning-Link (R) R-PE Kit (703-0010) (3) 43

Product General Protocols

FAQs for Lightning-Link (R) R-PE Kit (703-0010). (Showing 1 - 10 of 12 FAQs).

1. Can I label a peptide/protein/antibody other than IgG?
   • Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.As the protocols provided were optimized for labeling IgGs, we would recommend you adjust the amount of material you add to the Lighting-Link® vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is half the size of an antibody (about 80kD), add half as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10ug kits enable you to do this using small amounts of material and therefore at a low cost.
2. 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. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
3. Do my antibody and buffer fit the requirements?
   • Your antibody should be purified (affinity purification is preferred), as other molecules with free amine groups will interfere with the reaction, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 um filtration is a method of sterilization, not purification.Your biomolecule should also be in a suitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5 to 8.5 and not in any of the following: ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size your purchased (for example: a 3x100ug kit enable you to label 3 lots of 100ug antibody) and the volume added should also match (eg: 100ul), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).NOTE: The amount and volume of antibody recommended above are for all Lightning-Link• kits offered by Innova Biosciences, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page.
4. How many labels will bind to my biomolecule?
   • It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein). Traditional labelling techniques often have F/P ratios in the range of 4-7:1, and our comparative data for staining with antibodies labelled with Lightning Link shows similar staining intensity. The ratio of dye to antibody is only ever an average for the population of labelled molecules, as individual molecules may have different amount of dye incorporated into them.
5. How stable will my new conjugate be?
   • The Lightning-Link® chemistry joins the label to the antibody via a uni directional stable, covalent bond. The stability of your conjugate will therefore be dependent on your antibody and label of choice. In our in-house studies, conjugates made with our Lightning-Link® kits were fully active after more than 18 months' storage, undiluted, at 4 degrees C. Conjugates stored in 50% glycerol at -20 degrees C were found to be even more stable (several years). Please see below for advice on storage conditions. Please note that tandem dye stability is lower than the other dyes'. Tandem conjugates are fully stable for about 3 months at 4 degrees C.
6. What are the best storage conditions for my new conjugate?
   • TA new conjugate can be stored for 12-18 months at 4 degrees C as long as the antibody will tolerate storage at 4 degrees C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4 degrees C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20 degrees C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20 degrees C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using our LifeXtend HRP Conjugate Stabilizer/Diluent. Please note that the preservative sodium azide will inhibit HRP. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate.
7. What can I do if my antibody formulation does not fit the requirements?
   • If your antibody 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,...). 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 kit protocols to see the antibody amount/volume suitable for each kit.If your antibody is already purified but its concentration is too low, you can concentrate it by 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.
8. What is the difference between Lightning-Link® and Lightning-Link® Rapid?
   • The benefit of the Rapid kits is that the conjugates can be generated faster because the conjugation and quenching incubation times are shorter. The antibody/protein/peptide considerations are exactly the same, as is the high quality of the final conjugate. DyLight® dyes are only available in the Rapid format.
9. Can I use your lightning-link kits to label other proteins rather than antibodies?
   • Yes, the kits work by using free amines, so as long as you have that the kit should work.
10. I have 2 vials of pure lyophilized TLR1 antibody (catalog# AF1484) and I want to label the antibodies with PE Flourochromes. I am not sure how many kits to order from Lightning Antibody Labeling Kits and if any antibody purification or concentration kit is required.
    • For our PE Lightning Link Conjugation kits, I would recommend purchasing the kit 703-0010 (suitable for conjugating 3 x 60ug of antibody). This is the kit that can conjugate the closest amount of antibody to the full quantity you have per vial. The next size up is suitable for conjugating 600ug of antibody per reaction, but is priced much higher than the 60ug reactions. For your reference, the 600ug kits are available as 703-0015 (1 x 600ug) or 703-0003 (5 x 600ug).
11. Is trehalose accepatable in PBS for label with Lightning Link  R-PE
    • The trehalose should not affect any protein modification. Trehalose is a common component as lyophilised excipient and we do not expect that it will interfere with the conjugation.
12. I had discussions with technical experts about residual fluorescence of unreacted dye after protein modification using lightening link (PE). I have been repeatedly ensured that the un reacted dye looses fluorescence upon quenching. This has subsequently been determined to be untrue. Am I interpreting your answers correctly?
    • Yes you are correct. The quencher does not stop the fluorescence or block the label. It blocks the reactive amine group from being able to link to the free amines. The process in theory is called quenching because the label can no longer bind and, therefore, the unbound label (in most applications) will be washed away in the first wash step. We do not use this label kit for an antibody or protein in an application such as your cell labeling. If you want to use this kit in cell labeling you will want to do a separation from the unincorporated label. If you would like to use a different labeling chemistry or separate the unincorporated by size exclusion chromatography then you can contact our Custom service https://www.rndsystems.com/services/protein-services
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