Lightning-Link HRP Antibody Labeling Kit


Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0030]
Immunohistochemistry-Paraffin: Lightning-Link HRP Antibody Labeling Kit [701-0030]
Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0030]
Immunohistochemistry-Paraffin: Lightning-Link HRP Antibody Labeling Kit [701-0030] - Brown= CD20 labeled with HRP, Red= CD3 labeled with alkaline phosphatase. Mouse anti-human CD20 was conjugated to HRP using more

Product Details

Applications WB, IHC-P
Concentration is not relevant for this product. Please see the protocols for proper use of this product.

Order Details

Lightning-Link HRP Antibody Labeling Kit Summary

Lightning-Link® is an innovative technology that enables direct labeling of antibodies, proteins, peptides or other biomolecules for use in R&D applications.

The procedure is:
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.

Horseradish peroxidase is a 44kDa glycoprotein with 6 lysine residues which can be conjugated to labeled biomolecules using Lightning-Link®. The enzyme label can be visualized by chromogenic reactions; for example diaminobenzidine (DAB) in the presence of hydrogen peroxide (H202) is converted in to a water insoluble brown pigment. Other substrates which can be used to measure horseradish peroxidase activity include ABTS, TMB and TMBUS.

Kit Type
Antibody Labeling Kit

Packaging, Storage & Formulations

Store at -20C. Avoid freeze-thaw cycles.
This kit contains 3 x 10ug reactions.
Concentration is not relevant for this product. Please see the protocols for proper use of this product.


Read Publications using 701-0030.

Kit Components

  1. Glass vial(s) of Lightning-Link mix (1, 3 or 5 vials, depending on pack size)
  2. 1 vial of LL Modifier reagent
  3. 1 vial of LL Quencher reagent


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

For more information please check out these useful links!
Fluorescent Labels Poster
Antibody Labeling Guide
Antibody Purification Guide

Lightning Link® is a registered trademark of Innova Biosciences.


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 HRP Kit (701-0030)(65)

We have publications tested in 6 applications: ELISA, FLOW, IA, IB, IP, WB.

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Showing Publications 1 - 10 of 65. Show All 65 Publications.
Publications using 701-0030 Applications Species
Ghaffarian R, Roki N, Abouzeid A et al. Intra- and trans-cellular delivery of enzymes by direct conjugation with non-multivalent anti-ICAM molecules J Control Release 2016 Sep 28 [PMID:27473764] (WB) WB
Mohibi S, Srivastava S, Bele A et al. Acetylation of Mammalian ADA3 is Required for its Functional Roles in Histone Acetylation and Cell Proliferation Mol. Cell. Biol. 2016 Jul 11 [PMID:27402865] (WB) WB
Laura RP, Dong D, Reynolds WF, Maki RA. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation PLoS One 2016 Feb 18 [PMID:26890638] (WB) WB
Liao MC, Muratore CR, Gierahn TM et al. Single-Cell Detection of Secreted ABeta and sAPPa from Human IPSC-Derived Neurons and Astrocytes J. Neurosci. 2016 Feb 3 [PMID:26843653] (FLOW) FLOW
Mir RA, Bele A, Mirza S et al. A novel interaction of ECD protein with R2TP complex component RUVBL1 is required for the functional role of ECD in cell cycle progression Mol Cell Biol 2015 Dec 28 [PMID:26711270] (WB) WB
Ahn SB, Mohamedali A, Anand S et al. Characterisation of the interaction between heterodimeric alpha v beta 6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics. J Proteome Res 2014 [PMID:25318615]
Hu Z, Chang H, Ge M et al. Development of antigen capture ELISA for the quantification of EIAV p26 protein. Appl Microbiol Biotechnol. 2014 [PMID:25256618] (IA) IA
Praekelt U, Reissbrodt R, Kresse A et al. Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor. J Med Microbiol 2014 [PMID:25231626] (IA) IA
Debald M, Jin JP, Linke A et al. Calponin-h2: a potential serum marker for the early detection of human breast cancer?. Tumour Biol. 2014 [PMID:25099617] (IA) IA
Saresella M, Piancone F, Marventano I et al. A role for the TIM-3/GAL-9/BAT3 pathway in determining the clinical phenotype of multiple sclerosis. FASEB J. 2014 [PMID:25091272]
Show All 65 Publications.

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Product General Protocols

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

Video Protocols

WB Video Protocol

FAQs for Lightning-Link HRP Kit (701-0030). (Showing 1 - 10 of 11 FAQs).

  1. What type of volume can be run with a 5 by 1mg or 1 by 5mg vial of LL-HRP?
    • The optimal concentration range for the antibodies to be labeled is between 1.0-2.5 mg/mL.
  2. Our customer would like to know how to evaluate the amount of HRP conjugated successfully on antibodies?
    • The amount of HRP conjugated is usually determined by the molar ratio used in the conjugation reaction. This easy approach is possible because – provided the protocols are followed – Lightning-Link reactions are very efficient, resulting in very high recovery. HRP is about 1/4 the size of an antibody (approx. 40 kDa versus approx. 160 kDa). Therefore, if 100 ug antibody is added to 100 ug HRP, there will be an average of 4 HRP molecules bound to each antibody molecule, and if 400 ug antibody is added to 100 ug HRP, there will be an average of 1 HRP molecule bound to each antibody molecule (and so on).
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. Show All 11 FAQs.

Additional Lightning-Link HRP Products

Lightning-Link HRP 701-0030

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