Lightning-Link® HRP Antibody Labeling Kit

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Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0010]
Immunohistochemistry-Paraffin: Lightning-Link HRP Antibody Labeling Kit [701-0010]
Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0010]
Immunohistochemistry-Paraffin: Lightning-Link HRP Antibody Labeling Kit [701-0010] - 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 (See protocol for further information).

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.
Kit Type
Antibody Labeling Kit

Applications/Dilutions

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 can be used to label up to 400 ug of antibody, and is supplied in one vial.
Reviewed Applications
Read 5 Reviews rated 3.2
using
701-0010 in the following application:

Publications
Read Publications using 701-0010.

Packaging, Storage & Formulations

Storage
Store at -20C.

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!
Antibody Labeling Guide
Antibody Conjugation Illustrated Assay

This product is manufactured by Expedeon Inc. and distributed by Novus Biologicals.

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) HRP Kit (701-0010)(80)

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


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ELISA
(1)
FLOW
(1)
IA
(32)
IB
(4)
IP
(1)
WB
(5)
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Showing Publications 1 - 10 of 80. Show All 80 Publications.
Publications using 701-0010 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]
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]
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
Hansen NU, Willumsen N, Sand JM et al. Type VIII collagen is elevated in diseases associated with angiogenesis and vascular remodeling. Clin Biochem. 2016 [PMID: 27234597]
Show All 80 Publications.

Reviews for Lightning-Link (R) HRP Kit (701-0010) (5) 3.25

Average Rating: 3.2
(Based on 5 reviews)
We have 5 reviews tested in 1 species: Other.

Reviews using 701-0010:
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ELISA
(3)
WB
(1)
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Other
(4)
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Images Ratings Applications Species Date Details
ELISA Lightning-Link (R) HRP 701-0010
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5
reviewed by:
Ryan Holly
ELISA Other 08/17/2010
View

Summary

ApplicationELISA
Sample TestedDECPG:BSA
SpeciesOther

Primary Anitbody

Dilution RatioIncubation Dilution Buffer: 10mM PBS, Dilution Ratio: 1:5000, Incubation Time: 2 hours, Incubation Temp: 25 degrees Celsius

Details

Detection NotesPositive Control: DECPG:BSA, Negative Control: BSA, Wavelength: 450 nM
ELISA Plate DetailsELISA Detection Method: TMB Substrate, ELISA Sample Concentration: Stock Samples
ELISA Lightning-Link (R) HRP 701-0010
Enlarge
5
reviewed by:
Ryan Holly
ELISA Other 08/17/2010
View

Summary

ApplicationELISA
Sample TestedAnti-phosphoserine
SpeciesOther
CommentsQuick and easy conjugation of lighting link HRP to polyclonal antibody. This was used b/c I wanted to do a sandwich Elisa using the same antibodies so one needed to have and HRP conjugated to it. Worked perfect.

Primary Anitbody

Dilution RatioIncubation Dilution Buffer: PBS pH 7.4, Dilution Ratio: 1:5000, Incubation Time: 1 hour, Incubation Temp: 25 degrees Celsius

Details

Detection NotesPositive Control: BSA phosphoserine, Negative Control: BSA, Wavelength: 450 nM
ELISA Plate DetailsELISA Detection Method: TMB Substrate, ELISA Sample Concentration: stock

Comments

CommentsQuick and easy conjugation of lighting link HRP to polyclonal antibody. This was used b/c I wanted to do a sandwich Elisa using the same antibodies so one needed to have and HRP conjugated to it. Worked perfect.
  3
reviewed by:
Anonymous
WB Other 02/26/2010
View

Summary

ApplicationWestern Blot
Sample TestedPancreatic Cancer Cell protein extract, Sample Amount: 30 ug
SpeciesOther
CommentsThis was a trial of the labelling system - which appeared to work great!

Blocking

Blocking DetailsBlocking Buffer: 5% Milk blotto, Blocking Time: 1 hour, Blocking Temp: 24 degrees Celsius

Primary Anitbody

Dilution RatioDilution Ratio: 1/500, Incubation Dilution Buffer: 3% BSA/PBS/0.1% Tween 20, Incubation Time: overnight, Incubation Temp: 4 C

Details

Detection NotesDetection Method: ECL, Exposure Time: 10 min, Positive Cnt'l: 2nd Ab, HRP, Specific Bands: 57kDa, Non-Specific Bands: 37 120kDa

Comments

CommentsThis was a trial of the labelling system - which appeared to work great!
 
reviewed by:
12/31/1969
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  3
reviewed by:
Anonymous
ELISA Other 02/02/2009
View

Summary

ApplicationELISA
Sample TestedWheat Gluten
SpeciesOther
Lot427
CommentsIt's hard for me to rate the product because we were using it to label a previously untested antibody that we had in such small quantities that we couldn't label it any other way. The antibody actually did not work in the sandwich assay, but did work in an antigen down test, so I think the conjugation worked OK.

Primary Anitbody

Dilution RatioIncubation Dilution Buffer: PBS/BSA, Incubation Time: 1 hour, Incubation Temp: 25 degrees Celsius

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-gluten, Secondary Ab Dilution: 1:500 - 1:64000

Details

ELISA Plate DetailsELISA Sample Concentration: 2ug/ml

Comments

CommentsIt's hard for me to rate the product because we were using it to label a previously untested antibody that we had in such small quantities that we couldn't label it any other way. The antibody actually did not work in the sandwich assay, but did work in an antigen down test, so I think the conjugation worked OK.

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 (R) HRP Kit (701-0010). (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.

Other Available Formats

HRP 701-0010

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Recent Reviews

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Ryan Holly
08/17/2010
Application: ELISA
Species: Other

Ryan Holly
08/17/2010
Application: ELISA
Species: Other

 
Anonymous
02/26/2010
Application: WB
Species: Other