Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link®

• ICC

Lab

Immunocytochemistry - Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (AB269823)

Direct immunofluorescence - Lightning-Link® Alexa Fluor® 647 Anti-Cytokeratin 19 antibody [EP1580Y].

Direct immunofluorescent staining using Recombinant Anti-Cytokeratin 19 antibody [EP1580Y] - BSA and Azide free (ab195872) labelled with Alexa Fluor^® 647 Conjugation Kit (Fast) - Lightning-Link® (ab269823).

Lightning-Link® Alexa Fluor^® 647 Anti-Cytokeratin 19 [EP1580Y] conjugate was used to stain wild-type MCF7 cells (top panel) and A431 negative cells (bottom panel).

The cells were fixed with 4% paraformaldehyde (10 min) or 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-Cytokeratin 19 Alexa Fluor^® 647 conjugate (shown in red) or ab195872 (Anti-Cytokeratin 19 antibody, unlabelled control) overnight at +4°C. ab195872 treated cells only were incubated with ab150077 at 1/1000 dilution for 1 hour at room temperature (shown in green).

Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Schematic Diagram

Supplier Data

Schematic Diagram - Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (AB269823)

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 ab269823 protocol booklet for more details.

Learn more about our Lightning-Link® conjugation kits here

• ICC

PubMed

Immunocytochemistry - Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (AB269823)

Kim, Jin-Wook et al. used Alexa Fluor® 647 Conjugation Kit Lightning-Link® as part of examining alterations in metastatic capacity through cathepsin A by leptin. They used the kit to conjugate anti-LAMP2a antibody for use in immunocytochemistry.
Confocal microscopy images of negative control CHMp cells. (a) DAPI stained CHMp cells; (b) Negative control image of Alexa 488-conjugated secondary antibody. (c,d) Negative control image of Alexa 647 - conjugated LAMP2a antibodies. The white arrow-heads indicate the location of LAMP2a only. The images are captured under 40X confocal microscope.

Image from Kim et al., Int j. of mol. Sci., 21(23):8963; doi: 10.3390/ijms21238963. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• ICC

PubMed

Immunocytochemistry - Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (AB269823)

Alexa Fluor® 647 Conjugation Kit (Fast)

Image from Watts, Lotte P., et al., Elife, 9: e58020; doi: 10.7554/eLife.58020. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• ICC

Lab

Immunocytochemistry - Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (AB269823)

Direct immunofluorescence - Lightning-Link® Alexa Fluor® 647 Anti-EGFR antibody [E235].

Direct immunofluorescent staining using Recombinant Anti-EGFR antibody [E235] - BSA and Azide free (ab227459) labelled with Alexa Fluor^® 647 Conjugation Kit (Fast) - Lightning-Link® (ab269823).

Lightning-Link® Alexa Fluor^® 647 Anti-EGFR antibody [E235] conjugate was used to stain wild-type A431 cells (top panel) and MCF7 negative cells (bottom panel). The cells were fixed with 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-EGFR Alexa Fluor^® 647 conjugate (shown in red) or ab227459 (Anti-EGFR antibody, unlabelled control) overnight at +4°C. ab227459 treated cells only were incubated with ab150077 at 1/1000 dilution for 1 hour at room temperature (shown in green).

Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC

Lab

Immunocytochemistry - Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (AB269823)

Direct immunofluorescence - Lightning-Link® Alexa Fluor® 647 Anti-Vimentin antibody [EPR3776].

Direct immunofluorescent staining using Recombinant Anti-Vimentin antibody [EPR3776] - BSA and Azide free (ab193555) labelled with Alexa Fluor^® 647 Conjugation Kit (Fast) - Lightning-Link® (ab269823).

Lightning-Link^® Alexa Fluor^® 647 Anti-Vimentin antibody [EPR3776] conjugate was used to stain Vimentin-wild-type HAP1 cells (top panel) and Vimentin-knock-out HAP1cells (bottom panel). The cells were fixed with 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-Vimentin Alexa Fluor^® 647 (shown in red) or ab193555 (Anti-Vimentin antibody, unlabelled control) overnight at +4°C. ab193555 treated cells only were incubated with ab150077 at 1/1000 dilution for 1 hour at room temperature (shown in green).

Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Conjugation

PubMed

Conjugation - Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (AB269823)

Yuan, Yue, et al used Alexa Fluor 647 Conjugation Kit (Fast) - Lightning-Link (ab269823) as part of examining changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. They used the kit to conjugate Alexa Fluor 647 to anti-CD4 antibody (OKT4) for use in single-molecule super-resolution imaging.
Representative TIRF-STORM images, and selected magnified regions (insets) of cell-surface CD4 (green) and HIV p24 (magenta). Scale bar = 2 µm.

Image from Yuan et al., Viruses, 13(1):142. doi: 10.3390/v13010142. Reproduced under the Creative Commons license https://creativecommons.org/licenses

• Ie

PubMed

Immunoelectrophoresis - Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (AB269823)

Thiriet, Pierre-Emmanuel, et al used Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (ab269823) as part of rapid and accurate diagnosis of Acute kidney injury (AKI). They used the kit to conjugate Alexa Fluor® 647 to monoclonal anti-Lipocalin-2/NGAL antibody for use in the development of a microfluidic analytical device .
(a-c) Chip description and operation. (a) Presentation of the chip layout. Beads and reagents can be successively injected through the two inlets visible on the left. The device consists of three incubation lines upstream and three concentration lines downstream, at the end of which the beads are accumulated in clusters (shown here in red). (b) Illustration of a sandwich immunoassay used for detection of biomarkers. The analyte we aimed to detect was captured by the bead decorated with capture antibody (cAb) and detection was performed thanks to the fluorescently labeled detection antibody (dAb). (c) Presentation of the successive steps performed on-chip to operate the platform, namely, (1) beads' loading, (2) incubation with detection antibodies and (3) release from the incubation line, (4) clustering in the concentration region, and (5) discarding through the outlet. For the sake of clarity, the species bound to the beads and the electrically activated arrays of electrodes are indicated for each step. (d-f) On-chip incubation of Neutrophil Gelatinase-Associated Lipocalin (NGAL) biomarker. (d) Observation of the small beads' clusters (circled in pink) before and after 15 min of incubation. The fluorescence signal arose from the binding of dAb-NGAL complex to cAb-decorated beads dielectrically trapped in the regions upstream to the electrode line. Three NGAL concentrations were injected in separate experiments, namely, 1 ng/mL, 10 ng/mL, and 100 ng/mL. (f) Fluorescence signal as a function of the incubation time for different NGAL concentrations. After 15 min all concentrations provided a signal greater than the control experiment, consisting of an injection of a solution in absence of NGAL molecules. The error bars were obtained by measuring the fluorescent signal from 10 clusters.

Image from Thiriet, Pierre-Emmanuel, et al., Biosensors, 10(12):212; doi: 10.3390/bios10120212. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/