Atto 488 Conjugation Kit (Fast) - Lightning-Link®

• ICC

PubMed

Immunocytochemistry - Atto 488 Conjugation Kit (Fast) - Lightning-Link® (AB269896)

Eichwald, Catherine, et al used Atto 488 Conjugation Kit (Fast) - Lightning-Link^® (ab269896) as part of examining the distribution of acetylated-microtubules (MTs) with respect to viroplasms. They used the kit to conjugate Atto 488 to mouse monoclonal anti-alpha tubulin antibody for use in immunocytochemistry/immunofluorescence (ICC/IF).
At 6 hours post-infection, SA11-infected CV-1 cells [multiplicity of infection (MOI); 25 VFU/ml] were fixed with methanol and immunostained for detection of viroplasms in red (anti-NSP5 antibody followed by a secondary antibody conjugated to Alexa 594), acetylated-MTs in cyan (mouse mAb anti-acetylated alpha-tubulin followed by a secondary antibody conjugated to Alexa 647), MTs in green (mouse mAb anti-alpha tubulin directly conjugated to Atto 488 (green)) and nuclei in blue (DAPI). Z-stack images were acquired by high-resolution CLSM and subsequently, 3D-reconstructions were performed using surface and filament algorithms from the surpass model of the Imaris 7.0 software (Bitplane, Switzerland). 3D-reconstructions are visualized from the topside (A, B and C) and the bottom side (D, E and F) of the preparation. Images show viroplasms (from A to F), acetylated-MTs (A, B, D and E) and MTs (A, C, D and F). Scale bars are 5 μm.

Image from Eichwald, Catherine, et al., PloS one; 7(10): e47947. doi: 10.1371/journal.pone.0047947. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• Schematic Diagram

Supplier Data

Schematic Diagram - Atto 488 Conjugation Kit (Fast) - Lightning-Link® (AB269896)

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 ab269896 protocol booklet for more details.

Learn more about our Lightning-Link® conjugation kits here

• ICC

PubMed

Immunocytochemistry - Atto 488 Conjugation Kit (Fast) - Lightning-Link® (AB269896)

Simanjuntak, Yogy, et al used Atto 488 Conjugation Kit (Fast) - Lightning-Link^® (ab269896) as part of examining the mechanisms of viral invasiveness in Japanese encephalitis virus (JEV). They used the kit to conjugate Atto 488 to Japanese encephalitis virus (JEV) for use in immunocytochemistry.
BE(2)C cells were infected with fluorescently labeled JEV (Atto488) for 2 h at room temperature with or without anti-integrin β3 and anti-vimentin antibody (0.75 and 1.5 μg/ ml). Mouse immunoglobulin G (IgG) was used as antibody control (1.5 μg/ml). Cells with or without antibody (1.5 μg/ ml) were stained with Alexa Fluor 568 phalloidin for F-actin (red) and Hoechst for nuclei (blue). Confocal microscopy images (630× original magnification).

Image from Simanjuntak, Yogy, et al., Frontiers in microbiology 8:651; doi: 10.3389/fmicb.2017.00651. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• IHC-Fr

PubMed

Immunohistochemistry (Frozen sections) - Atto 488 Conjugation Kit (Fast) - Lightning-Link® (AB269896)

Veldkamp, Marieke W., et al used Atto 488 Conjugation Kit (Fast) - Lightning-Link^® (ab269896) as part of examining electrophysiological effects of the neuropeptide Sub-P in isolated rabbit atrial myocytes, and in Langendorff-perfused and in situ rabbit heart. They used the kit to conjugate Atto 488 to anti-NK3R antibody for use in immunohistochemistry (frozen sections).
Sections of rabbit (upper panel) and human (lower panel) left atrium showing labelling of the NK-3R (green) in the peripheral sarcolemma of cardiomyocytes, co-stained for the intracellular myocardial marker a-actinin (red). Scale bar 25 μM.

Image from Veldkamp, Marieke W., et al., Nature communications, 9(1):4357. doi: 10.1038/s41467-018-06530-5. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• Flow Cyt

PubMed

Flow Cytometry - Atto 488 Conjugation Kit (Fast) - Lightning-Link® (AB269896)

Flow Cytometry - Atto 488 Conjugation Kit (Fast) - Lightning-Link.

Robinson, Andrew P., et al used Atto 488 Conjugation Kit (Fast) - Lightning-Link^® (ab269896) as part of characterizing oligodendroglial populations. They used the kit to conjugate Atto 488 to Rat anti-PDGFRalpha antibody, clone APA5, for use in flow cytometry.
SJL/J mice were immunized with PLP139–151 and scored daily for clinical disease. A cohort of SJL/J mice was sacrificed, and spinal cords were analyzed by flow cytometry (n = 5). (A) Cells were distinguished from debris by forward and side scatter then singlet cells were gated. Live cells were gated by dead cell exclusion, and CNS resident cells were identified as CD45− or CD45low. (B) Oligodendroglial cells were defined by double positive staining : A2B5+PDGFRα+ early OPCs, A2B5+NG2+ intermediate OPCs, NG2+O4+ late OPCs, O4+MOG+ pre-myelinating oligodendrocytes, and GALC+MOG+ mature oligodendrocytes.

Image from Robinson, Andrew P., et al., PloS one, 9(9):e107649. doi: 10.1371/journal.pone.0107649. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• Conjugation

PubMed

Conjugation - Atto 488 Conjugation Kit (Fast) - Lightning-Link® (AB269896)

Atto 488 Conjugation Kit (Fast) - Lightning-Link® labeling TcdA1–1874 toxin for Flow cytometry.

Olling A et al. used ab269896 as part of examining the role of toxins in the pathogenicity of Clostridium difficile.

They used the kit to conjugate Atto 488 to TcdA1–1874 toxin for use in flow cytometry.

Binding of fluorescent labeled TcdA-PE/Cy5 and TcdA1–1874-Atto488 to HT29 and CHO-C6 cells was investigated by FACS analysis. Right shift of the black curve illustrates toxin binding which was detected through fluorescence emission at 667 nm for TcdA and at 523 nm for TcdA1–1874, respectively. Due to different ratio of fluorophor and toxin, fluorescence intensity of TcdA-PE/Cy5 cannot directly be compared with TcdA1–1874-Atto488.

Image from Olling A et al., PLoS One, 6(3):e17623. Fig 5.; doi: 10.1371/journal.pone.0017623. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• Conjugation

PubMed

Conjugation - Atto 488 Conjugation Kit (Fast) - Lightning-Link® (AB269896)

Atto 488 Conjugation Kit (Fast) - Lightning-Link® labeling p17 protein for Microscopy.

Zeinolabediny Y et al. used ab269896 as part of examining the toxicity of p17 protein assemblies.

They used the kit to conjugate Atto 488 to p17 protein for use in microscopy.

Representative images of the p17 localisation as overlays of Atto 488-fluorescence and light microscopy in worms.

Scale bar = 50 μm. Worms fed for 2 h vehicle (10 mM PB, pH 7.4) or 4 nM p17-Atto 488.

Image from Zeinolabediny Y et al., Sci Rep., 7(1):10313. Fig 3.; doi: 10.1038/s41598-017-10875-0. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/