Lightning-Link® Type A Biotin Antibody Labeling Kit

Product Discontinued

Lightning-Link® Type A Biotin Antibody Labeling Kit Summary

• Description: Lightning-Link is an innovative technology that enables direct labeling of proteins, peptides or other biomolecules.
  
  Key Features:
  
  • 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.
  
  The attachment of biotin to biomolecules is an important laboratory technique. Biotin binds to the tetrameric avidin proteins, including streptavidin and neutravidin, with exceptionally high affinity, and this interaction is exploited in various applications such as Western Blot, Immunohisthochemistry and ELISA. Our biotinylation kits are optimised to create high quality conjugates and minimise assay background. There is also no need for a wash or desalting step following the biotinylation reaction, saving you both time and material.
  
  Lightning-Link Biotin has been optimised for two separate applications:
  • Type A - intended for assays in which a streptavidin-labeled detection reagent will be used.
  • Type B - optimised for assays in which the biotinylated protein is captured by streptavidin immobilized on a surface (ie plates, nitrocellulose, magnetic beads etc).
• Marker: Optimized for Streptavidin-Labeled Detection
• Kit Type: Antibody Labeling Kit

Applications/Dilutions

• Dilutions:
  • Immunohistochemistry
  • Immunomicroscopy
  • Immunoprecipitation
• 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 20 ug of antibody.

Packaging, Storage & Formulations

• Storage: Store at -20C. Avoid freeze-thaw cycles.
• Buffer: This kit contains 3 x 10ug reactions.

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.

Background

Why Lightning-Link® Biotin?

The attachment of biotin to biomolecules is an important laboratory technique. Biotin binds to the tetrameric avidin proteins, including streptavidin and neutravidin, with exceptionally high affinity, and this interaction is exploited in various applications such as western blotting, immunohisthochemistry and ELISA.

Our biotinylation kits are optimised to create high quality conjugates and minimise assay background. There is also no need for a wash or desalting step following the biotinylation reaction, saving you both time and material.


Lightning-Link® Biotin has been optimised for two separate applications:

Type A - intended for assays in which a streptavidin-labeled detection reagent will be used,
Type B - optimised for assays in which the biotinylated protein is captured by streptavidin immobilized on a surface (ie plates, nitrocellulose, magnetic beads etc).

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) Type A Biotin Kit (704-0030)(27)

Publications using 704-0030

• Ashby EL, Miners JS, Kumar S et al. Investigation of A beta phosphorylated at serine 8 (pA beta ) in Alzheimer's disease, dementia with Lewy bodies and vascular dementia. Neuropathol Appl Neurobiol 2014-01-01 [PMID: 25556395] (IA)
• Grabias B, Zheng H, Mlambo G et al. A sensitive enhanced chemiluminescent-ELISA for the detection of Plasmodium falciparum circumsporozoite antigen in midguts of Anopheles stephensi mosquitoes. J Microbiol Methods 2014-01-01 [PMID: 25455023] (IA)
• Lu Z, Rynkiewicz MJ, Madico G et al. GroEL of Francisella tularensis. PLoS One 2014-01-01 [PMID: 24968190] (IA)
• Skinner C, Patfield S, Stanker LH et al. New High-Affinity Monoclonal Antibodies against Shiga Toxin 1 Facilitate the Detection of Hybrid Stx1/Stx2 In Vivo. PLoS One 2014-01-01 [PMID: 24914553] (IA)
• Miners JS, Jones R, Love S. Differential Changes in A beta 42 and A beta 40 with Age. J Alzheimers Dis 2014-01-01 [PMID: 24503618]
• Seite JF, Goutsmedt C, Youinou P et al. Intravenous immunoglobulin induces a functional silencing program similar to anergy in human B cells. J Allergy Clin Imm 2013-01-01 [PMID: 24139609]
• Skinner C, Patfield S, Stanker L, He X. Development of Monoclonal Antibodies and Immunoassays for Sensitive and Specific Detection of Shiga Toxin Stx2f. PLoS One 2013-01-01 [PMID: 24069462] (IA)
• Naidu VG, Dinesh Babu KR, Thwin MM et al. RANKL targeted peptides inhibit osteoclastogenesis and attenuate adjuvant induced arthritis by inhibiting NF- kappa B activation and down regulating inflammatory cytokines. Chem Biol Interact 2013-01-01 [PMID: 23333834] (IA)
• He X, McMahon S, Skinner C et al. Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk. J Immunol Methods. 2012-01-01 [PMID: 23279946]
• Ferrara F, Naranjo LA, Kumar S et al. Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker. PLoS One 2012-01-01 [PMID: 23166701] (IA)
• Liu M, Zhao L, Gao Y et al. Epitope recognition patterns of thyroglobulin antibody in sera from patients with Hashimoto's thyroiditis on different thyroid functional status. Clin Exp Immunol. 2012-01-01 [PMID: 23121669] (IA)
• Zhang Y, Chen YC, Krummel MF, Rosen SD. Autotaxin through Lysophosphatidic Acid Stimulates Polarization, Motility, and Transendothelial Migration of Naive T Cells J Immunol 2012-10-15 [PMID: 22962684]
• Chiu LL, Reis LA, Momen A, Radisic M. Controlled release of thymosin beta 4 from injected collagen-chitosan hydrogels promotes angiogenesis and prevents tissue loss after myocardial infarction. Regen Med 2012-01-01 [PMID: 22817626]
• Spencer JD, Hains DS, Porter E et al. Human Alpha Defensin 5 Expression in the Human Kidney and Urinary Tract. PLoS One 2011-01-01 [PMID: 22359618] (IA)
• Spencer JD, Schwaderer AL, Dirosario JD et al. Ribonuclease 7 is a potent antimicrobial peptide within the human urinary tract. Kidney Int 2011-01-01 [PMID: 21525852] (Primate)
• Morgan R, Boxall A, Bhatt A et al. Engrailed-2 (EN2): A Tumor Specific Urinary Biomarker for the Early Diagnosis of Prostate Cancer. Clin Cancer Res 2011-01-01 [PMID: 21364037] (IA)
• Gijsen M, King P, Perera T et al. HER2 Phosphorylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Herceptin in Breast Cancer. PLoS Biol. 2010-01-01 [PMID: 21203579] (IP)
• Frederick JR, Fitzpatrick JR 3rd, McCormick RC et al. Stromal Cell-Derived Factor-1alpha Activation of Tissue-Engineered Endothelial Progenitor Cell Matrix Enhances Ventricular Function After Myocardial Infarction by Inducing Neovasculogenesis. Circulation. 2010-09-14 [PMID: 20837901]
• Charbonneau NL, Carlson EJ, Tufa S et al. In Vivo Studies of Mutant Fibrillin-1 Microfibrils. J Biol Chem 2010-01-01 [PMID: 20529844] (IA)
• Tanriver Y, Ratnasothy K, Bucy RP et al. Targeting MHC Class I Monomers to Dendritic Cells Inhibits the Indirect Pathway of Allorecognition and the Production of IgG Alloantibodies Leading to Long-Term Allograft Survival. J Immunol. 2010-01-01 [PMID: 20083658]
• Borai A, Livingstone C, Ghayour-Mobarhan M et al. Serum insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation status in subjects with and without ischaemic heart disease. Artherosclerosis 2010-01-01 [PMID: 19732896] (IA)
• Sarugaser R, Hanoun L, Keating A et al. Human Mesenchymal Stem Cells Self-Renew and Differentiate According to a Deterministic Hierarchy. PLoS One 2009-01-01 [PMID: 19652709] (IF/IHC)
• Al-Dujaili EA, Mullins LJ, Bailey MA, Kenyon CJ. Development of a Highly Sensitive ELISA for Aldosterone in Mouse Urine: Validation in Physiological Pathophysiological States of Aldosterone Excess Depletion - . Steroids. 2008-01-01 [PMID: 19162057]
• Bao S, Wu Q, Li Z et al. Targeting Cancer Stem Cells through L1CAM Suppresses Glioma Growth. Cancer Res 2008-01-01 [PMID: 18676824]
• Marr AK, Jenssen H, Moniri MR et al. Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1. Biochemie 2009-01-01 [PMID: 18573311]
• Velappan N, Clements J, Kiss C et al. Fluorescence linked immunosorbant assays using microtiter plates. J Immunol Methods. 2013-01-01 [PMID: 18514691]
• Bossart KN, Tachedjian M, McEachern JA et al. Functional studies of host-specific ephrin-B ligands as Henipavirus receptors. Virology 2008-01-01 [PMID: 18054977] (IA)

FAQs for Lightning-Link (R) Type A Biotin Kit (704-0030). (Showing 1 - 8 of 8 FAQs).

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.