What functional groups do I need on my protein?

Lightning-Link requires amine groups on the molecule to be labeled. Most proteins have lysine and/or alpha-amino groups. All antibodies will have multiple amine functions.

Do I need to purify the conjugate?

No. The chemicals used in Lightning-Link are deactivated by the quencher, and the by-products are benign and do not need to be removed.

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 (approximately 40 kDa versus approximately 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).

Can non-antibody molecules be labeled?

Yes. While labeling of antibodies is a major application, the only requirement is that the protein to be labeled has amine functionality.

I am interested in the antibody labeling kit and was wondering if it would work for proteins (not antibodies) as well. Specifically, I'm looking to see if it's possible to conjugate either biotin or streptavidin to LAP (the TGF-B1 surrogate).

Yes they can be used for many types of biomolecules: Lightning-Link® technology works by targeting amine groups and can therefore be used for most biomolecules including antibodies, proteins and peptides. Please see the FAQs section of this protocol for recommended adjustments for biomolecules that differ in size from IgGs, including proteins and peptides.

Do you know if the conjugation affects the binding capability of the proteins (such as to antibodies, or vice versa)?

The binding of the label does not affect the confirmation of the antibody and, therefore, will not inhibit the antibody binding to the target. The label binds to amine groups but does not inhibit the active site.

Lightning-Link (R) HRP Antibody Labeling Kit

Name: Lightning-Link (R) HRP Antibody Labeling Kit
Brand: Novus Biologicals
SKU: 701-0000
Rating: 5 (5 reviews)

Novus Biologicals, part of Bio-Techne | Catalog # 701-0000

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Specific Notices

Key Product Details

Applications

Functional Assay, Immunohistochemistry, Western Blot

Kit Type

Antibody Labeling Kit

Conjugate

HRP

Product Summary for Lightning-Link (R) HRP Antibody Labeling Kit

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 FAQs

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

Product Specifications

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 with 3 vials, each suitable for labeling up to 400 ug of antibody.

Scientific Data Images for Lightning-Link (R) HRP Antibody Labeling Kit

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]

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Kit Contents for Lightning-Link (R) HRP Antibody Labeling Kit

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

Formulation, Preparation, and Storage

Concentration

Concentration is not relevant for this product. Please see the protocols for proper use of this product.

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store at -20C.

Background: Lightning-Link (R) HRP

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%.

Additional Lightning-Link (R) HRP Products

Product Documents for Lightning-Link (R) HRP Antibody Labeling Kit

Product Datasheets

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Protocols & Manuals

Download Manual - Lightning-Link (R) HRP Antibody Labeling Kit

COA

SDS

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Product Specific Notices for Lightning-Link (R) HRP Antibody Labeling Kit

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.

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.

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