Recombinant trimeric SARS-CoV-2 spike recombinant protein (AcroBiosystems) was labeled with the FITC Conjugation Kit (Fast)-Lightning-Link (Abcam), according to the manufacturer’s instructions. Unlabeled protein was detected by means of fixation/permeabilization (BD Biosciences) and anti–spike 1 (S1) (GeneTex) and anti-rabbit APC (BioLegend) antibodies. ACE2 binding was blocked with 2-µg/mL anti-ACE2 (R&D Systems) for 3 hour before spike recombinant protein addition [7 (link)]. Cells were treated with 2.5 µg/mL recombinant protein, and cell viability was verified using the MTT assay (Sigma-Aldrich; not shown).
Fitc conjugation kit fast lightning link
Manufactured by Abcam
Sourced in United Kingdom
The FITC Conjugation Kit (Fast)-Lightning-Link is a lab equipment product designed for the conjugation of proteins with the fluorescent dye FITC (Fluorescein Isothiocyanate). The kit provides a fast and convenient method to label proteins with FITC without the need for specialized equipment or extensive purification steps.
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Lab products found in correlation
Anti ace2, by R&D Systems (1 mentions)
Mtt assay, by Merck Group (1 mentions)
P9416 100ml, by Merck Group (1 mentions)
P4417 100tab, by Merck Group (1 mentions)
Stabletemp, by Cole-Parmer (1 mentions)
Vascular permeability assay kit, by Merck Group (1 mentions)
Recombinant mouse il 1β, by Thermo Fisher Scientific (1 mentions)
4 protocols using fitc conjugation kit fast lightning link
1
SARS-CoV-2 Spike Protein Binding Assay
2
Lateral Flow Assay for Cervical Cancer Detection
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Nitrocellulose membrane (1UN18ER100025NT)
was purchased from Sartorius (Göttingen, Germany). Aqueous
suspension of single-layer GO (S1319112702) was purchased from Global
Graphene Group (Dayton, OH, USA). According to the supplier, this
2D material displays an average lateral size around 50 nm and its
C/O ratio is around 1. Tween 20 (P9416-100ML) and phosphate buffer
saline (PBS) tablets (P4417-100TAB) were purchased from Sigma-Aldrich
(Saint Louis, MI, USA). FITC Conjugation Kit Fast-Lightning-Link (ab188285)
was purchased from Abcam (Cambridge, UK). According to previously
reported procedures,11 (link),18 (link) anti-SLD monoclonal antibody
and SLD peptide were produced by our team at the Universidad Autónoma
de Guerrero (Chilpancingo, Guerrero, Mexico). Vaginal swab samples
were collected by “Servicio de Diagnóstico Integral
en la detección Oportuna del Cancer Cérvico Uterino”
at Universidad Autónoma de Guerrero (Chilpancingo, Guerrero,
Mexico). Signed consent was obtained from those women who participated
in this research. Wax patterns were printed using a ColorQube 8085
from Xerox (Stanford, CT, USA). A hot plate StableTemp from Cole-parmer
(Vernon Hills, IL, US) was used to heat and fabricate the paper-based
devices. All the fluorescent micrographs were acquired through a Cytation
5 multimodal reader from BioTek (Winooski, VT, USA).
was purchased from Sartorius (Göttingen, Germany). Aqueous
suspension of single-layer GO (S1319112702) was purchased from Global
Graphene Group (Dayton, OH, USA). According to the supplier, this
2D material displays an average lateral size around 50 nm and its
C/O ratio is around 1. Tween 20 (P9416-100ML) and phosphate buffer
saline (PBS) tablets (P4417-100TAB) were purchased from Sigma-Aldrich
(Saint Louis, MI, USA). FITC Conjugation Kit Fast-Lightning-Link (ab188285)
was purchased from Abcam (Cambridge, UK). According to previously
reported procedures,11 (link),18 (link) anti-SLD monoclonal antibody
and SLD peptide were produced by our team at the Universidad Autónoma
de Guerrero (Chilpancingo, Guerrero, Mexico). Vaginal swab samples
were collected by “Servicio de Diagnóstico Integral
en la detección Oportuna del Cancer Cérvico Uterino”
at Universidad Autónoma de Guerrero (Chilpancingo, Guerrero,
Mexico). Signed consent was obtained from those women who participated
in this research. Wax patterns were printed using a ColorQube 8085
from Xerox (Stanford, CT, USA). A hot plate StableTemp from Cole-parmer
(Vernon Hills, IL, US) was used to heat and fabricate the paper-based
devices. All the fluorescent micrographs were acquired through a Cytation
5 multimodal reader from BioTek (Winooski, VT, USA).
3
Assessing Lung Endothelial Permeability
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Lung primary microvascular endothelial cells (Cell Biologics, C57-6011) were grown in phenol red–free endothelial cell medium (ScienCell), seeded in collagen-coated inserts, and permeability was determined using a vascular permeability assay kit according to the manufacturer’s instructions (Millipore). FITC-conjugated anti-COLV antibody, labeled using a FITC Conjugation Kit (Fast) - Lightning-Link (Abcam), was added to wells in the absence or presence of 50 ng/mL recombinant mouse IL-1β (Thermo Fisher Scientific) and fluorescence was red with a plate reader with filters appropriate for 485 nm and 535 nm excitation and emission, respectively.
4
Fluorescent Antigen Conjugation Protocol
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In order to study uptake kinetics, we made use of fluorescently labeled antigens conjugated to non-fluorescent particles. The antigens were fluorescently labeled using a FITC Conjugation Kit (Fast)—Lightning-Link® (Abcam, Cambridge, UK). The assay was performed according to the manufacturer’s protocol to yield 1 mg of labeled antigens. Hereafter, the FITC-labeled antigens were linked to 12.5 mg of non-fluorescent 3 µm and 500 nm amino-functionalized polystyrene particles using the method as described above for the non-labeled antigens. To every condition, 400 µg of FITC-labeled antigens (either influenza antigens or HBsAg) was added. Hereafter, the conjugation efficiency was determined with a modified Pierce™ Coomassie Plus Assay (Thermo Fisher Scientific). Conjugation was visually confirmed by microscopy, using a Deltavision™ Elite high-resolution fluorescence microscope (GE Healthcare UK Ltd., Little Chalfont, UK) equipped with a 60× oil immersion objective. Data acquisition was performed using the system-integrated GFP/mCherry filter setting in combination with the Deltavision SoftWoRx™ 6 acquisition and deconvolution software (GE Healthcare). The data were subsequently analyzed and processed using FIJI imaging software [35 (link)].
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