Product namePE/Cy5.5® Conjugation Kit - Lightning-Link®
PE/Cy5.5® Conjugation Kit / PE/CY5.5® Labeling Kit ab102871 uses a simple and quick process for PE/Cy5.5 labeling / conjugation of antibodies. It can also be used to conjugate other proteins or peptides. Learn about our antibody labeling kits and their advantages.
To conjugate an antibody to PE/Cy5.5® using this kit:
- add modifier to antibody and incubate for 3 hrs
- add quencher and incubate for 30 mins
The conjugated antibody can be used immediately in WB, ELISA, IHC etc. No further purification is required and 100% of the antibody is recovered for use.
Learn about buffer compatibility below; for incompatible buffers and low antibody concentrations, use our rapid antibody purification and concentration kits. Use the FAQ to learn more about the technology, or about conjugating other proteins and peptides to PE/Cy5.5®.
Custom size conjugation kits up to 100 mg are available on demand. Please contact us to discuss your requirements.
This product is manufactured by Expedeon, an Abcam company, and was previously called Lightning-Link® R-PE/Cy5.5 Labeling Kit. 761-0005 is the same as the 60 µg size. 761-0010 is the same as the 3 x 60 ug size. 761-0030 is the same as the 3 x 10 ug size. 761-0015 is the same as the 600 µg size.
Amount and volume of antibody for conjugation to PE/Cy5.5®.
Kit size Recommended
amount of antibody
3 x 10 µg 3 x 10 µg 3 x 10 µL 60 µg 1 x 60 µg 1 x 60 µL 3 x 60 µg 3 x 60 µg 3 x 60 µL 600 µg 1 x 600 µg 1 x 600 µL
1Ideal antibody concentration is 1mg/ml. 0.5 - 1 mg/ml can be used if the maximum antibody volume is not exceeded. Antibodies > 1 mg/ml or < 0.5 mg/ml should be diluted /concentrated.
The selling size of this product has been changed – it is now based on the amount of antibody that can be conjugated with the kit, not the amount of PE mix provided. The amount of antibody advised that can be used with the kit has also been updated to reflect what will give the best conjugation results. The quantity and formulation of reagents provided have not changed, if you have been previously using the kit successfully with a different amount of antibody, there is no need to change the way that you are using the kit.
Buffer Requirements for Conjugation
Buffer should be pH 6.5-8.5.
Compatible buffer constituents
If a concentration is shown, then the constituent should be no more than the concentration shown. If several constituents are close to the limit of acceptable concentration, then this can inhibit conjugation.
50mM / 0.6% Tris1 0.1% BSA 50% glycerol 0.1% sodium azide PBS Potassium phosphate Sodium chloride HEPES Sucrose Sodium citrate EDTA Trehalose
1 Tris buffered saline is almost always ≤ 50 mM / 0.6%
Incompatible buffer constituents
Thiomerosal Proclin Glycine Arginine Glutathione DTT
Only purified antibodies are suitable for use, ie. where other proteins, peptides, or amino acids are not present: antibodies in ascites fluid, serum or hybridoma culture media are incompatible.
Storing and handling conjugation kits
Lyophilized Lightning-Link® components are hygroscopic.
Kits are intentionally shipped at ambient temperature with silica gel to avoid exposure to moisture. Upon receipt, store the kit frozen and protect from moisture. Before opening the outer container, allow the lyophilized components to reach room temperature to minimize condensation.
Storage instructionsStore at -20°C. Please refer to protocols.
Components 60 µg 600 µg 3 x 10 µg 3 x 60 µg ab274106 - Modifier reagent 1 x 200µl 1 x 200µl 1 x 200µl 1 x 200µl ab274147 - PE/Cy5.5 mix 1 x 60µg 1 x 600µg 3 x 10µg 3 x 60µg ab274133 - Quencher reagent 1 x 200µl 1 x 200µl 1 x 200µl 1 x 200µl
Sancey, Lucie, et al used PE/Cy5.5® Conjugation Kit - Lightning-Link® (ab102899) as part of examining AGuIX NP uptake measured by FCM as a function of time in the 3 OncoCilAir™ subpopulations. They used the kit to conjugate PE/Cy5.5® to IgG isotype control antibody for use in flow cytometry.
a Summary of AGuIX® uptake with the percentage of AGuIX®+ cells and their corresponding MFI values presented after 24 and 72 h of NP exposure (n=6/conditions, from 3 distinct batches of cell culture with and without mucus). The relative uptake ratios were calculated from Eq. 3. Statistical analysis was performed using the Mann-Whitney test (*p 0.05 compared to GFP-negative cells). The results b-i were obtained from samples of the 2nd series of cell cultures, presenting a fast tumor growth and an intense AGuIX® uptake at T24h. FACS plots of AGuIX® uptake (% of AGuIX+-cells and geometric mean MFI) according to the 3 gates (b, f) that discriminate GFP-negative (red channel; c, g), GFP+ (green channel; d, h) and GFP++ (blue channel; e, i) tumor cells at 24 h (b-e) and 72 h (f-i)
This illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.
Wang, Shuyan, et al used PE/Cy5.5® Conjugation Kit - Lightning-Link® (ab102899) as part of examining Neph3-positive cells co-cultured with rat cerebellar slices. They used the kit to conjugate PE/Cy5.5® to anti-Neph3 monoclonal antibody for use in flow cytometry.
(A) Schematic representation of sorting and co-culture procedures. (B, C) FACS analysis of neural rosette cells on Day 20 of differentiation. (D) Immunofluorescence staining to confirm that the sorted cells were Neph3/GFP-double positive. Bar = 100 µm. (E) The sorted cells were co-cultured with cerebellum slices and examined at 1 week and 4 weeks. Bar = 100 µm.
ab102899 has been referenced in 6 publications.
- Pasciuto E et al. Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition. Cell 182:625-640.e24 (2020). PubMed: 32702313
- Sancey L et al. Multiparametric investigation of non functionalized-AGuIX nanoparticles in 3D human airway epithelium models demonstrates preferential targeting of tumor cells. J Nanobiotechnology 18:129 (2020). PubMed: 32912214
- Syeda MM et al. Prediction of breast cancer risk based on flow-variant analysis of circulating peripheral blood B cells. Genet Med 19:1071-1077 (2017). PubMed: 28301456
- Wang S et al. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons. Sci Rep 5:9232 (2015). PubMed: 25782665
- Hesdorffer CS et al. Distinctive immunoregulatory effects of adenosine on T cells of older humans. FASEB J 26:1301-10 (2012). PubMed: 22121051
- Carter CC et al. HIV-1 infects multipotent progenitor cells causing cell death and establishing latent cellular reservoirs. Nat Med 16:446-51 (2010). PubMed: 20208541