Lightning-Link HRP Antibody Labeling Kit

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Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0000]
Immunohistochemistry: Lightning-Link HRP Antibody Labeling Kit [701-0000]
Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0000]
Immunohistochemistry: Lightning-Link HRP Antibody Labeling Kit [701-0000] - Brown= CD20 labeled with HRP, Red= CD3 labeled with alkaline phosphatase. Mouse anti-human CD20 was conjugated to HRP using ...read more

Product Details

Summary
Conjugate
HRP
Format
HRP
Kit Type
Antibody Labeling Kit

Order Details

Lightning-Link HRP 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.

Our HRP antibody labeling kit enables the direct conjugation of Horseradish Peroxidase to any biomolecule with an available amine group. The researcher simply pipettes their antibody or other biomolecule into the vial of Lightning-Link HRP and incubates for 3 hours.

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


Horseradish peroxidase is a 44kDa glycoprotein with 6 lysine residues. 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.

Popular conjugates applications:
1. Western Blot
2. Immunohistochemistry
3. ELISA
Kit Type
Antibody Labeling Kit

Applications/Dilutions

Reviewed Applications
Read 5 Reviews rated 5
using
701-0000 in the following application:

Publications
Read Publications using
701-0000 in the following applications:

  • 2 publications
  • IA
    32 publications
  • IB
    4 publications
  • IP
    1 publication
  • WB
    5 publications

Packaging, Storage & Formulations

Storage
Store at -20C. Avoid freeze-thaw cycles.

Kit Components

Components
  1. 1 vial of LL-Modifier reagent
  2. 1 vial of LL-Quencher reagent
  3. 1 or 3 or 5 glass vial(s) of Lightning-Link mix

Notes

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.

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 HRP Kit (701-0000)(86)

We have publications tested in 1 confirmed species: Mouse.

We have publications tested in 5 applications: ELISA, IA, IB, IP, WB.


Filter By Application
ELISA
(2)
IA
(32)
IB
(4)
IP
(1)
WB
(5)
All Applications
Filter By Species
Mouse
(1)
All Species
Showing Publications 1 - 10 of 86. Show All 86 Publications.
Publications using 701-0000 Applications Species
Welch NG, Lebot CJ, Easton CD et al. Polypropylene microtitre plates modified with [Cr(OH)6]3 - for enhanced ELISA sensitivity. J Immunol Methods. 2017 [PMID: 28365327]
Hijmans RS, Rasmussen DG, Yazdani S et al. Urinary collagen degradation products as early markers of progressive renal fibrosis. J Transl Med. 2017 Mar 20 [PMID: 28320405]
Boi S, Dis EV, Hansen EJ et al. Latent murine leukemia virus infection characterized by the release of non-infectious virions. Virology. Mar 11 2017 [PMID: 28292718]
Phares TW, May AD, Genito CJ et al. Rhesus macaque and mouse models for down-selecting circumsporozoite protein based malaria vaccines differ significantly in immunogenicity and functional outcomes. Malar. J. Mar 13 2017 [PMID: 28288639] (ELISA, Mouse) ELISA Mouse
Rasmussen DG, Sand JM, Karsdal MA, Genovese F. Development of a Novel Enzyme-Linked Immunosorbent Assay Targeting a Neo-Epitope Generated by Cathepsin-Mediated Turnover of Type III Collagen and Its Application in Chronic Obstructive Pulmonary Disease. PLoS One. 2017 Jan 11 [PMID: 28076408]
Furman JL, Vaquer-Alicea J, White CL 3rd et al. Widespread tau seeding activity at early Braak stages. Acta Neuropathol. 2017 [PMID: 27878366]
Welch NG, Madiona RM, Easton CD et al. Chromium functionalized diglyme plasma polymer coating enhances enzyme-linked immunosorbent assay performance. Biointerphases. 2016 [PMID: 27835921]
Welch NG, Easton CD, Scoble JA et al. A chemiluminescent sandwich ELISA enhancement method using a chromium (III) coordination complex. J Immunol Methods. 2016 [PMID: 27650427]
Welch NG, Scoble JA, Easton CD et al. High-throughput Production of Chromium (III) Complexes for Antibody Immobilization. Anal Chem. 2016 [PMID: 27644116]
Mao S, Ou X, Zhu D et al. Development and evaluation of indirect ELISAs for the detection of IgG, IgM and IgA1 against duck hepatitis A virus 1. J Virol Methods. 2016 [PMID: 27577105]
Show All 86 Publications.

Reviews for Lightning-Link HRP Kit (701-0000) (5) 55

Average Rating: 5
(Based on 5 reviews)
We have 5 reviews tested in 2 species: Human, Other.

Reviews using 701-0000:
Filter by Applications
ELISA
(3)
WB
(1)
IHC-P
(1)
All Applications
Filter by Species
Human
(2)
Other
(2)
All Species
Images Ratings Applications Species Date Details
 
reviewed by:
Anonymous
ELISA 09/04/2014
View

Summary

ApplicationELISA
 
reviewed by:
Anonymous
ELISA Human 06/03/2011
View

Summary

ApplicationELISA
Sample TestedCustom made peptide
SpeciesHuman
CommentsI conjugated my first antibody with HRP for direct ELISA couple of times, excellent results

Primary Anitbody

Dilution RatioIncubation Dilution Buffer: PBS, Dilution Ratio: 1:1000, Incubation Time: 2 hours, Incubation Temp: 37 degrees Celsius

Details

Detection NotesPositive Control: concentration curve, Negative Control: PBS, Wavelength: 370
ELISA Plate DetailsELISA Detection Method: TMB, ELISA Sample Concentration: 0.1 to 2000 ng/ml

Comments

CommentsI conjugated my first antibody with HRP for direct ELISA couple of times, excellent results
Western Blot Lightning-Link HRP 701-0000
Enlarge
reviewed by:
Robert Hnasko
WB Other 04/18/2011
View

Summary

ApplicationWestern Blot
Sample Testedrecombinant protein, Sample Amount: 100ng
SpeciesOther
CommentsFast, efficient, stable direct HRP conjugation - best reagent we have found - works as described to label antibodies useful for WB and ELISA</p><p>

Blocking

Blocking DetailsBlocking Buffer: 10% NFDM in TBST, Blocking Time: 1 hour, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1ug/mL, Incubation Dilution Buffer: TBST + 0.05% IgG free BSA, Incubation Time: 30 min, Incubation Temp: RT

Details

Detection NotesDetection Method: ECL, Exposure Time: 20 seconds, Positive Control: recombinant, Specific Bands: Yes, Non-Specific Bands: No

Comments

CommentsFast, efficient, stable direct HRP conjugation - best reagent we have found - works as described to label antibodies useful for WB and ELISA</p><p>
 
reviewed by:
Anonymous
ELISA Other 04/02/2009
View

Summary

ApplicationELISA
Sample TestedPlasma protein
SpeciesOther
Lot636
CommentsVery good kit! This kit was used for a Sandwich ELISA assay.

Primary Anitbody

Dilution RatioIncubation Dilution Buffer: BSA based Hepes, Incubation Time: 30 minutes, Incubation Temp: 24 degrees Celsius

Secondary Antibody

Secondary DescriptionSecondary Ab: Mouse Monoclonal HRP

Comments

CommentsVery good kit! This kit was used for a Sandwich ELISA assay.
 
reviewed by:
Anonymous
IHC-P Human 02/19/2009
View

Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedHuman
SpeciesHuman
Lot528
CommentsThis conjugation kit worked beautifully. Procedure is so simple. We are satisfied by the final staining result. I had 0.1% sodium azide in original anti-mouse antibody (it is purified IgG fraction). Based on the product instruction, 0.1% sodium azide is upper limit that this conjugation kit can tolerate. Although we had some concern, it worked well. However, we still exchanged buffer to remove it after conjugation, so it would inhibit HRP activity.

Blocking

Blocking DetailsBlocking Buffer: Normal horse serum, Blocking Time: 30 minutes, Blocking Temp: 20 degrees Celsius

Primary Anitbody

Dilution RatioIncubation Dilution Buffer: PBS, Incubation Time: 30 minutes, Incubation Temp: 20 degrees Celsius

Secondary Antibody

Secondary DescriptionSecondary Ab: Goat anti-mouse IgG, Secondary Ab Dilution: Undiluted

Comments

CommentsThis conjugation kit worked beautifully. Procedure is so simple. We are satisfied by the final staining result. I had 0.1% sodium azide in original anti-mouse antibody (it is purified IgG fraction). Based on the product instruction, 0.1% sodium azide is upper limit that this conjugation kit can tolerate. Although we had some concern, it worked well. However, we still exchanged buffer to remove it after conjugation, so it would inhibit HRP activity.

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 (701-0000). (Showing 1 - 10 of 13 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 13 FAQs.

Additional Lightning-Link HRP Products

Lightning-Link HRP 701-0000

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Recent Reviews

5
5
5
4
0
3
0
2
0
1
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Anonymous
09/04/2014
Application: ELISA
Species:

 
Anonymous
06/03/2011
Application: ELISA
Species: Human

Robert Hnasko
04/18/2011
Application: WB
Species: Other