Lightning-Link® FluoProbes647H Antibody Labeling Kit Summary
Lightning-Link is an innovative technology that enables direct labeling of proteins, peptides or other biomolecules.Key Features:
- Easy to use
- Requires 30 sec hands-on time
- No spin or separation steps involved
The researcher simply pipettes the antibody or other biomolecule into a vial of lyophilized mixture containing the label of interest and incubates for either 3 hours or See Lighning-Link Rapid for only 15 min incubation.
Despite its apparent simplicity, the Lightning-Link process is sophisticated and generates conjugates with performance characteristics identical to, or better than, those prepared with laborious multistep conjugation procedures.
Antibody Labeling Kit
By circumventing the desalting or dialysis steps that commonly interrupt traditional antibody conjugation procedures, LightningLink technology can be used to label both small (e.g. 10 ug) and large quantities of primary antibodies with ease. Batch-to-batch variation upon scale up is minimal as the process is so simple, and recoveries are always 100%. This kit is supplied with 3 vials, each suitable for labeling up to 200 ug of antibody.
Packaging, Storage & Formulations
Store at -20C. Avoid freeze-thaw cycles.
- 1 or 3 glass vial(s) of Lightning-Link mix
- 1 vial of LL-Quencher reagent
- 1 vial of LL-Modifier reagent
Alternate Names for Lightning-Link (R) FluoProbes647H Antibody Labeling Kit
- Lightning conjugation kits
- Lightning link conjugation kits
- Lightning Link
- Lightning-link conjugation kits
Easy FluoProbes647H labeling of your antibody with 30 seconds hands-on time. FluoProbes(R)647H is one of a new generation of fluorescent labels designed for labelling biomolecules. It has a strong absorption at 653nm, high fluorescence at 674nm (extinction coefficient 2.5 x10^5 cm^-1M^-1) and high quantum yield. The Lightning-Link conjugation system represents a quantum leap forward in conjugation technology. It allows you to make antibody conjugates with minimal hands-on time - less than 30 seconds. Lightning-Link simplifies immunoassay techniques, such as western blotting, ELISA and immunohistochemistry, by eliminating secondary reagents and cutting the number of incubation and wash steps.
Despite its simplicity, Lightning-Link is a very sophisticated conjugation system in which the antibody is directionally coupled to the label (and not to itself) in a controlled fashion, creating high quality conjugates. As the procedure is not interrupted by desalting steps, trial conjugates can be prepared with microgram quantities of protein and then scaled up with ease. Lightning-Link eliminates problems associated with scale up; hands-on time is essentially independent of process scale. Consistency from batch to batch is easy to achieve, and recoveries approach 100%.
This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed
for 6 months from date of receipt.
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Product General Protocols
View specific protocols for Lightning-Link (R) FluoProbes647H Kit (791-0010):
Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.
FAQs for Lightning-Link (R) FluoProbes647H Kit (791-0010). (Showing 1 - 10 of 10 FAQ).
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.
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.
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.
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.
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.
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.
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.
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.
I am going to use Lightning-Link FluoProbes 647H conjugation kit to measure phagosomal pH by FACS. I wonder if I can dissolve the lyophilized mixture in DMSO?
- The Lightning-Link vial does not simply contain dye, but also other chemicals. We do not know how well all the components will dissolve or perform in DMSO.
I don't want to label antibody with this kit but I am going to label 3um amino beads with FITC + Flu probes 647H, now which concentration I should use to label my beads?
- The Lightning-Link kits work by targeting available amine groups on the molecule to be labeled, they are not antibody-specific. However, as the kits have been optimized for use with antibodies, we cannot specify the amount of beads to add to form the best conjugate - this would have to be determined by experimentation. We would therefore suggest adding a different amount of beads to several glass vials; that way, you could obtain a degree of optimization with minimal cost, material and time. Normally we suggest a 3 x 10 ug pack size is used for this, but if you wanted to you could use your 3 x 100 ug kit. Please note are that the usual Lightning-Link rules will still apply, e.g. your beads should be in a suitable volume, and be free from other substances that would interfere in the conjugation e.g. other amines. You can refer to the protocols, guides and FAQ on our website for more information.
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