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. | Features | Benefits | | Quick and easy to use | Save time, no special knowledge required | | No separation steps | 100% recovery - no antibody/protein loss | | Can be used in a wide range of applications | Flexible | | Freeze dried | Ships at ambient temperature, long shelf-life | | Fully scalable (10 ug to 1 g or more) | Easy transfer from R&D to manufacturing | | Stringently QC tested | Consistent high quality, excellent batch-to-batch reproducibility | | Large number of labels available | Experimental flexibility | | Reliable: nearly 300 references | Successfully 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. Learn more about Lightning-Link™ Conjugation Kits by reading FAQsFor more information please check out these useful links! Antibody Labeling Guide Antibody Conjugation Illustrated Assay |
| Kit Type |
Antibody Labeling Kit |
Applications/Dilutions
| Dilutions |
- Immunohistochemistry
- Immunohistochemistry-Paraffin
- Western Blot
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| 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 4 mg of antibody, and is supplied in one vial. |
| Reviewed Applications |
Read 2 Reviews rated 4.5 using
701-0002 in the following applications:
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Packaging, Storage & Formulations
Kit Components
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Components
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- 1 or 3 or 5 glass vial(s) of Lightning-Link mix
- 1 vial of LL-Modifier reagent
- 1 vial of LL-Quencher reagent
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Notes
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
Easy HRP labeling of your antibody with 30 seconds hands-on time. Antibodies are labeled to enzymes in order to amplify the signal via the catalytic properties of the enzyme. Horseradish peroxidase (HRP) labeling is one of the most commonly used enzymes for this purpose. HRP conjugates are ideal for use in ELISA, immunoblotting and immunohistochemistry applications. HRP has a faster catalytic rate than alkaline phosphatase (AP) and is more sensitive. 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%.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%.
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.
Product General Protocols
Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.
Video Protocols
FAQs for Lightning-Link (R) HRP Kit (701-0002). (Showing 1 - 10 of 11 FAQs).
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What type of volume can be run with a 5 by 1mg or 1 by 5mg vial of LL-HRP? ..show answer..
- The optimal concentration range for the antibodies to be labeled is between 1.0-2.5 mg/mL.
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Our customer would like to know how to evaluate the amount of HRP conjugated successfully on antibodies? ..show answer..
- 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).
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Can I label a peptide/protein/antibody other than IgG? ..show answer..
- 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.
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Do I need a wash or desalt step? ..show answer..
- 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.
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Do my antibody and buffer fit the requirements? ..show answer..
- 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.
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How many labels will bind to my biomolecule? ..show answer..
- 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.
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How stable will my new conjugate be? ..show answer..
- 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.
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What are the best storage conditions for my new conjugate? ..show answer..
- 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.
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What can I do if my antibody formulation does not fit the requirements? ..show answer..
- 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.
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What is the difference between Lightning-Link® and Lightning-Link® Rapid? ..show answer..
- 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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Can I use your lightning-link kits to label other proteins rather than antibodies? ..show answer..
- Yes, the kits work by using free amines, so as long as you have that the kit should work.
Show All 11 FAQs.
Additional Lightning-Link (R) HRP Products
Blogs on Lightning-Link (R) HRP
![Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0002]](http://images.novusbio.com/fullsize/Lightning-Link-HRP-Antibody-Labeling-Kit-Western-Blot-701-0002-img0003.jpg "Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0002]")
![Immunohistochemistry-Paraffin: Lightning-Link HRP Antibody Labeling Kit [701-0002]](http://images.novusbio.com/fullsize/Lightning-Link-HRP-Antibody-Labeling-Kit-Immunohistochemistry-Paraffin-701-0002-img0006.jpg "Immunohistochemistry-Paraffin: Lightning-Link HRP Antibody Labeling Kit [701-0002]")
![Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0002]](http://images.novusbio.com/fullsize/Lightning-Link-HRP-Antibody-Labeling-Kit-Western-Blot-701-0002-img0002.jpg "Western Blot: Lightning-Link HRP Antibody Labeling Kit [701-0002]")
![Immunohistochemistry-Paraffin: Lightning-Link HRP Antibody Labeling Kit [701-0002] - Brown= CD20 labeled with HRP, Red= CD3 labeled with alkaline phosphatase. Mouse anti-human CD20 was conjugated to HRP using Lightning-Link® kits, and rat anti human CD3 was conjugated to Alkaline Phosphatase using Lightning Link®. Both antibodies were then used simultaneously to stain formalin fixed paraffin fixed embedded sections of human lymph node. CD20 positive cells are visualized with DAB and are stained brown, and CD3 visualized with Fast Red and stained red.](http://images.novusbio.com/fullsize/Lightning-Link-HRP-Antibody-Labeling-Kit-Immunohistochemistry-Paraffin-701-0002-img0005.jpg "Immunohistochemistry-Paraffin: Lightning-Link HRP Antibody Labeling Kit [701-0002] - Brown= CD20 labeled with HRP, Red= CD3 labeled with alkaline phosphatase. Mouse anti-human CD20 was conjugated to HRP using Lightning-Link® kits, and rat anti human CD3 was conjugated to Alkaline Phosphatase using Lightning Link®. Both antibodies were then used simultaneously to stain formalin fixed paraffin fixed embedded sections of human lymph node. CD20 positive cells are visualized with DAB and are stained brown, and CD3 visualized with Fast Red and stained red.")
