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Antibody Labeling Kits

Lightning-Link® - for labeling primary antibodies, proteins or peptides

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Primary antibody and protein labeling

 

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

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 (Lightning-Link range) or only 15 min (Lightning-Link Rapid range).

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.

To learn more, read our introductory guide to antibody labeling.

Use our protein calculator to find out more about labeling proteins other than antibodies.

Contact us to find out more:

 

 
 

Manufacturing

The technology is fully scalable (?g to grams) ensuring quality and consistency without any deterioration in the performance of the conjugate. As there are no separation or desalting steps, antibody recovery is 100%. Furthermore, the bond between the label and biomolecule is covalent, therefore the conjugate is extremely stable.

 
  • "We’ve been really impressed with Innova’s products. Their biotin kit is simple to use and proved more durable than many competitors’ kits, and we’ve seen significant improvements in reproducibility since we started to use them regularly. We’re looking forward to continuing to work with the Lightning–Link® technology.”

    Dr Jake Micallef, CSO of VolitionRx

  •  

  • "Like many other small diagnostics companies we have used our own HRP activation and conjugation techniques for some years. However, on trying Innova’s Lightning Link kit we found its simplicity and the high level of functionality of the conjugates produced to be significantly better.

    We have since tried another, similar kit but the resulting product was decidedly inferior, with lower signal and higher noise. We plan to continue using Lightning–Link® for our existing ELISAs and also for others to be added to our food testing range in the next few years.”

    Chief Executive at Bio-Check (UK) Limited

  •  

  • See what others have said here!
Lightning-Link® conjugates are used in a large number of scientific applications including:
We also have a range of educational videos available, please follow the links below:
 

Which label should I use?

You can consult our table containing the specifications of all our Lightning-Link® fluorescent dyes. You can view our enzyme and other labels on the Lightning-Link page. You can also view our products categorised by application here.

There are also datasheets (including absorption/emission spectra) available on the individual web page for each fluorescent product - see the Lightning-Link page for a list.

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.

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 optimised 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 ½ the size of an antibody (about 80kD), add ½ as much to the vial. Please note this is only a guideline and the best amount for your assay should be determined experimentally; our 3x10µg kits enable you to do this using small amounts of material and therefore at a low cost.

Finally, please note the usual Lighting-Link® requirements will still apply (see below).

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 µm filtration is a method of sterilisation, 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 3x100µg kit enable you to label 3 lots of 100µg antibody) and the volume added should also match (eg: 100µl), meaning the ideal concentration is of 1mg/ml. For other biomolecule, the amount added should be adjusted by changing your concentration (see above).

*Use our concentration calculator to help you calculate the amount of biomolecule to add to a Lightning-Link kit.*

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.

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.

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 optimised 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.

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.

What about enzymes and fluorescent proteins?

For most of the products, the number of labels per biomolecule is outlined in the individual protocols. If it is not listed, please contact our technical team at technical.enquiries@innovabiosciences

How stable will my new conjugate be?

The Lightning-Link® chemistry joins the label to the antibody via a uni dirrectional stable, covalent bond. The stability of your conjugate will therefore be dependant 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°C. Conjugates stored in 50% glycerol at -20°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°C.

What are the best storage conditions for my new conjugate?

A new conjugate can be stored for 12-18 months at 4°C as long as the antibody will tolerate storage at 4°C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4°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°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°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.

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.

 

 
Lightning-Link ProductsClick the individual products for further information, pricing and data.

Enzyme Labeling Kits

Biotin / Streptavidin Labeling Kits

Fluorescence Labeling Kits

Lightning-Link®*
Lightning-Link® Rapid*
Maximal Absorbance
(nm)
Maximal Emission
(nm)
DyLight® 350
 
353
432
AMCA
 
346
442
Atto 390
 
390
479
DyLight® 405
 
400
420
PerCP 718-0030 482 677
PerCP/Cy5.5 763-0030 482 700
PE/Cy5 760-0030 488 670
DyLight® 488
 
493
518
Fluorescein
494
520
Atto488
501
523
R-Phycoerythrin 703-0030 535 575
PE/Texas Red® 767-0030 535 615
PE/Atto594 768-0030 535 627
PE/Cy5.5 761-0030 535 694
PE/Cy7 762-0030 535 776
Rhodamine
544
576
B-Phycoerythrin 716-0005 546 575
Cyanine Dye 3 (Cy3)
550
570
DyLight® 550
 
562
576
Atto 565
 
563
592
FluoProbes594
 
591
616
DyLight® 594
 
593
618
Texas Red®
 
595
615
Atto 633
 
629
657
APC/Cy5.5 764-0030 633 694
APC/Cy7 765-0030 633 776
DyLight® 633
 
638
658
Cyanine Dye 5 (Cy5)
643
667
Allophycocyanin 705-0005 650 662
DyLight® 650
 
652
672
FluoProbes647H
653
675
Cyanine Dye 5.5 (Cy5.5)
 
675
694
DyLight® 680
 
682
715
Atto700
 
700
725
DyLight® 755
 
754
776
DyLight® 800
 
770
794
* The antibody labeling kits come in two formats - Lightning-Link® and Lightning-Link® Rapid, with incubation    times of 3 hours and 15 minutes respectively.

by InnovaBiosciences 




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