Lightning-Link® APC-Cy7 Antibody Labeling Kit

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Flow Cytometry: Lightning-Link APC-Cy7 Antibody Labeling Kit [765-0010]

Product Details

Summary
Conjugate
Allophycocyanin/Cy7
Format
Allophycocyanin/Cy7 (A=650;755, E=767)
Kit Type
Antibody Labeling Kit

Order Details

Lightning-Link® APC-Cy7 Antibody Labeling Kit Summary

Description
Lightning-Link antibody labeling kits enable the direct labeling of antibodies, proteins, peptides or other biomolecules for use in R&D applications, drug discovery and the development of diagnostic kits (See protocol for further information).

The easy-to-use, one step procedure allows researchers to covalently label biomolecules with only 30 seconds hands on time. The researcher simply pipettes the biomolecule into a vial of lyophilized mixture containing the label of interest and incubates.

FeaturesBenefits
Quick and easy to useSave time, no special knowledge required
No separation steps100% recovery - no antibody/protein loss
Can be used in a wide range of applicationsFlexible
Freeze driedShips at ambient temperature, long shelf-life
Fully scalable (10 ug to 1 g or more)Easy transfer from R&D to manufacturing
Stringently QC testedConsistent high quality, excellent batch-to-batch reproducibility
Large number of labels available Experimental flexibility
Reliable: nearly 300 referencesSuccessfully used in many fields of research


APC/Cy7 is a tandem conjugate. The APC is excited at 652nm and functions as an energy donor for the Cy7. Energy is transferred from the APC to the Cy7 via FRET (Fluorescence Resonance Energy Transfer). The Cy7 emits the energy received from the APC in the form of long wavelength light at 790nm
Kit Type
Antibody Labeling Kit

Applications/Dilutions

Dilutions
  • Flow Cytometry
Application Notes
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 as 3 vials.
Reviewed Applications
Read 2 Reviews rated 5
using
765-0010 in the following application:

Publications
Read Publications using 765-0010.

Packaging, Storage & Formulations

Storage
Store at -20C.

Kit Components

Components
  1. 1 or 3 glass vial(s) of Lightning-Link mix
  2. 1 vial of LL-Modifier reagent
  3. 1 vial of LL-Quencher reagent

Notes

Learn more about Lightning-Link™ Conjugation Kits by reading FAQs

For more information please check out these useful links!
Antibody Labeling Guide
Antibody Conjugation Illustrated Assay

This product is manufactured by Abcam and distributed by Novus Biologicals.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. This product is guaranteed for 1 year from date of receipt and this statement overrides any mentioned guarantee period on the limitations section of this products datasheet. Please contact technical@novusbio.com with questions.

Background

Lightning-Link is a one step antibody labeling kit, requiring just 30 seconds hands-on time. The antibody is covalently bonded to the label in a directional and controlled process at near-neutral pH.Label - APC/Cy7 is a tandem conjugate. The APC is excited at 633nm and functions as an energy donor for the Cy7. Energy is transferred from the APC to the Cy7 via FRET (Fluorescence Resonance Energy Transfer). The Cy7 emits the energy received from the APC in the form of long wavelength light at 767nm.Features:- 30 seconds hands-on time- No separation steps- No losses- Scalable technology

Limitations

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.

Publications for Lightning-Link (R) APC-Cy7 Kit (765-0010)(8)

We have publications tested in 1 application: FLOW.


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FLOW
(6)
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Showing Publications 1 - 8 of 8.
Publications using 765-0010 Applications Species
Calenda G, Villegas G, Reis A et al. Mucosal susceptibility to HIV infection in the proliferative and secretory phases of the menstrual cycle. AIDS Res. Hum. Retroviruses Jan 2 2019 [PMID: 30600686]
Okagawa T, Konnai S, Nishimori A et al. Bovine immunoinhibitory receptors contribute to the suppression of Mycobacterium avium subsp.paratuberculosis-specific T-cell responses Infect Immun. 2015 Oct 19 [PMID: 26483406] (FLOW) FLOW
Freeman CM, Crudgington S et al. Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS). J Transl Med 2015 [PMID: 25622723] (FLOW) FLOW
Robinson AP, Rodgers JM, Goings GE, Miller SD. Characterization of Oligodendroglial Populations in Mouse Demyelinating Disease Using Flow Cytometry: Clues for MS Pathogenesis. PLoS One 2014 [PMID: 25247590] (FLOW) FLOW
Taichman RS, Patel LR, Bedenis R et al. GAS6 Receptor Status Is Associated with Dormancy and Bone Metastatic Tumor Formation. PLoS One 2013 [PMID: 23637920] (FLOW) FLOW
Robinson AP, Caldis MW, Harp CT et al. High-mobility group box 1 protein (HMGB1) neutralization ameliorates experimental autoimmune encephalomyelitis J Autoimmun 2013 Mar 17 [PMID: 23514872]
Jones CL, Ferreira S, McKenzie RC et al. Regulation of T-Plastin Expression by Promoter Hypomethylation in Primary Cutaneous T-Cell Lymphoma. J Invest Dermatol 2013 [PMID: 22495182] (FLOW) FLOW
Kim N, Kukkonen S, Gupta S et al. Association of Tat with Promoters of PTEN and PP2A Subunits Is Key to Transcriptional Activation of Apoptotic Pathways in HIV-Infected CD4+ T Cells PLoS Pathogens 2010 [PMID: 20862322] (FLOW) FLOW

Reviews for Lightning-Link (R) APC-Cy7 Kit (765-0010) (2) 52

Average Rating: 5
(Based on 2 reviews)

Reviews using 765-0010:
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(1)
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Images Ratings Applications Species Date Details
  5
reviewed by:
Vincent Giacalone
ICC/IF 06/19/2018
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Summary

ApplicationImmunocytochemistry/Immunofluorescence
LotIBS4124

Comments

CommentsWe used this labeling kit to conjugate EGFR antibody, which we then used for flow cytometry. These kits are reliable and easy to use, and very helpful for designing panels if you cannot find an antibody with the fluorochrome you need
  5
reviewed by:
Vincent Giacalone
06/19/2018
View

Summary

ApplicationSerum
LotIBS4124

Comments

CommentsWe used this labeling kit to conjugate EGFR antibody, which we then used for flow cytometry. These kits are reliable and easy to use, and very helpful for designing panels if you cannot find an antibody with the fluorochrome you need

Product General Protocols

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

FAQs for Lightning-Link (R) APC-Cy7 Kit (765-0010). (Showing 1 - 9 of 9 FAQs).

  1. Could you please recommend methods to assess the degree of conjugation (using your Lightning conjugation kit. APC or APC Cy7 or other fluorophore)?
    • For the Lightning Link conjugation kits, there is no way for us to measure the ratio of conjugate per mg of antibody. The conjugate label binds to all free amines. The conjugation process has an efficiency rate of approximately 95%. As these are kits that we distribute for Innova Biosciences, this is all the data I have regarding efficiency for these labeling kits. If you have any further questions, please let me know and I will contact Innova on your behalf to see if they have any additional data to provide.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.

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Vincent Giacalone
06/19/2018
Application: ICC/IF
Species:

 
Vincent Giacalone
06/19/2018
Application:
Species: