Cy5® Conjugation Kit (Fast) - Lightning-Link®

• ICC

PubMed

Immunocytochemistry - Cy5® Conjugation Kit (Fast) - Lightning-Link® (AB188288)

Lee, Yoon Seon, et al used Cy5® Conjugation Kit (Fast) - Lightning-Link® (ab188288) as part of examining the translocation and localization of newly developed peptide derived from CPNE7 (Cpne7-DP) . They used the kit to conjugate Cy5® to Cpne7-DP oligopeptide for use in immunocytochemistry.
The intracellular distribution of Cpne7-DP is shown after odontoblastic MDPC-23 cells were treated with Cy5-labeled Cpne7-DP (10 μg/mL).

Image from Lee et al., Materials, 13(20):4618; doi: 10.3390/ma13204618. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• Schematic Diagram

Supplier Data

Schematic Diagram - Cy5® Conjugation Kit (Fast) - Lightning-Link® (AB188288)

This illustration demonstrates a general procedure of how the Lightning-Link®(Fast) labeling technology enables the direct labeling of antibodies or proteins.

Simply pipette your antibody or biomolecule of choice into the vial of a lyophilized mixture containing the label of interest and incubate for just 15 minutes. Please see the ab188288 protocol booklet for more details.

Learn more about our Lightning-Link® conjugation kits here

• Fluorescence Microscopy

PubMed

Fluorescence Microscopy - Cy5® Conjugation Kit (Fast) - Lightning-Link® (AB188288)

Li, Tiankuan, et al used Cy5® Conjugation Kit (Fast) - Lightning-Link® (ab188288) as part of examining immunotherapeutical treatments for lung cancer. They used the kit to conjugate Cy5® to anti-PD-L1 mAb for use in fluorescence microscopy.
CLSM images of docetaxel and CY5-labelled anti-PD-L1 mAb-co-loaded microbubbles with a shell containing FITC, scale bar = 20 μm.

Image from Li, Tiankuan, et al., Biomaterials science, 8(5):1418-1430. doi: 10.1039/c9bm01575b. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/3.0/

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Cy5® Conjugation Kit (Fast) - Lightning-Link® (AB188288)

Cy5® Conjugation Kit - Lightning-Link® labeling NK2A peptide for confocal microscopy.

Dassanayake, Rohana P et al used ab188288 as part of examining the antimicrobial activity of bovine NK-lysin-derived peptides.

They used the kit to conjugate Cy5® to NK2A peptide for use in confocal microscopy.

H. somni was incubated with unconjugated Cy5 (A) or 20 μM NK2A-Cy5 conjugate for 30 min and bacterial nuclear contents were stained with DAPI. NK2A-Cy5 conjugate is shown in red and DAPI is shown in blue.

Image from Dassanayake RP et al., PloS One, 12(8):e0183610. Fig 4.; doi:10.1371/journal.pone.0183610. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• Flow Cyt

PubMed

Flow Cytometry - Cy5® Conjugation Kit (Fast) - Lightning-Link® (AB188288)

Lindenburg, Laurens, et al used Cy5® Conjugation Kit (Fast) - Lightning-Link® (ab188288) as part of examining microemulsion-based bead display screening of the ZIgE SpliMLiB library. They used the kit to conjugate Cy5® to Native human IgE protein (Azide free) (ab65866) for use in FACS.
(A) Schematic overview of a round of SpliMLiB-enabled directed evolution of ZIgE. SpliMLiB beads (i) were singly encapsulated in emulsion IVTT at 37°C for 1 h (ii), sufficient time to allow for both ZIgE-SpyCatcher variants' expression as well as for their SpyTag-SpyCatcher-mediated immobilisation on the bead surface, after which the emulsion was broken, and the washed beads were exposed to Cy5-labelled IgE (iii), followed by flow cytometric sorting of beads based on Cy5 signal (iv). (B) Representative histogram recorded during the flow cytometric sorting of SpliMLiB ZIgE library beads. The range of fluorescence intensity used for each of the sorting gates 1-4 is indicated. (C) Analysis of pooled, recovered and subcloned DNA from the sorting gates set out in panel B. DNA was used to express protein in IVTT under bulk, i.e. non-emulsion conditions, in the presence of SpyTag-functionalised microbeads. The microbeads, having captured the SpyCatcher fusion proteins, were then incubated with 200 nM IgE-Cy5 and analysed by flow cytometry. Cy5 fluorescence intensity was normalised to a sample prepared from beads exposed to purified ZIgEwild-type-SpyCatcher protein (WT, grey bar). Negative control (NC) was beads not exposed to any ZIgE-SpyCatcher protein. (D) Analysis of bacterially expressed & purified variants derived from the stringently sorted library output from FACS sorting gate 4. Beads that had been bound with ZIgE-SpyCatcher variants were incubated with 200 nM IgE-Cy5 and analysed by flow cytometry. ZIgEwild-type-SpyCatcher (labelled WT) was included as control and was used to normalise all fluorescent values. The variant showing the highest Cy5 median signal (variant 33, marked by a single asterisk) and second highest (variant 44, marked by a double asterisk) signal were taken forward for further analysis. (E) As panel D, except for 48 randomly picked clones derived from the unsorted SpliMLiB input library beads. (F) Frequencies of amino acids encountered in selected variants displaying a higher binding signal than ZIgEwild-type-SpyCatcher (17 in total). The most frequent amino acid at each position is indicated in bold to emphasise it.

Image from Lindenburg, Laurens, et al., Nucleic acids res., 8(11): e63; doi: 10.1093/nar/gkaa270. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• WB

PubMed

Western blot - Cy5® Conjugation Kit (Fast) - Lightning-Link® (AB188288)

Mody, Karishma T., et al used Cy5^® Conjugation Kit (Fast) - Lightning-Link^® (ab188288) as part of examining the integrity of Bm86 protein postlabeling. They used the kit to conjugate Cy5^® to R. micr-protein ladder, lane 5-Cy5-Bm86 protein, lane 7-Cy5-Bm86/ Rho-SV-140-C18 Supernatant, lane 8-Cy5-Bm86/Rho-SV-140-C18 pellet (first antibody 869 polyclonal sheep 1/5,000; second antibody monoclonal anti-goat/sheep 1/10,000). The Cy5-labeled Bm86 retained its native antigenicity as it was recognized by the antibodies in serum from a sheep immunized with the unlabeled antigen from a previous study.

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Image from Mody et al., Front bioeng biotechnol., 8:606652; doi: 10.3389/fbioe.2020.606652. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• FRET

PubMed

FRET - Cy5® Conjugation Kit (Fast) - Lightning-Link® (AB188288)

Mellal, Katia, et al used Cy5^® Conjugation Kit (Fast) - Lightning-Link^® (ab188288) as part of examining effects of MPE-001 on the CD36/TLR2 interaction. They used the kit to conjugate Cy5® to anti-CD36 antibody (ab80080) for use in FRET.
Peritoneal MPs were stimulated with 300 ng/ml R-FSL1 in the presence of 10-7 M MPE-001 or vehicle. (A) MPE-001 disrupted the interaction between CD36 labeled with Cy5 (red) using the Cy5® Conjugation Kit (Fast) - Lightning-Link® (ab188288) and TLR2 labeled with Cy3 (green) using the Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as assessed by FRET after 5 min stimulation with R-FSL1. (B) Percentage of energy transfer measured using LSM-700 confocal microscope (Zeiss). One-way ANOVA test with Newman-Keuls post-test for multiple comparison was performed. *P<0.05, **P<0.01 and ***P <0.001 vs R-FSL1. Data are shown as mean ±S.E.M.

Image from Mellal et al., Sci rep., 9(1):12903. doi: 10.1038/s41598-019-49472-8. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/