Alexa fluor 405 nhs ester

Manufactured by Thermo Fisher Scientific

Alexa Fluor 405 NHS Ester is a fluorescent labeling reagent. It is designed for conjugation to primary amines on proteins and other biomolecules. The product emits fluorescence in the blue-violet region of the visible spectrum.

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9 protocols using alexa fluor 405 nhs ester

Fluorescent Labeling of Lectins

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Lectins were labelled with Alexa Fluor 405 NHS Ester, Alexa Fluor 488 NHS Ester (Molecular Probes) or Cy5 NHS Ester (GE Healthcare) according to the manufacturer’s protocol for amine-reactive probes. Free dye was removed using Zeba Spin desalting columns with 7K MWCO (Thermo Fisher Scientific).

Villringer S., Madl J., Sych T., Manner C., Imberty A, & Römer W. (2018). Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion. Scientific Reports, 8, 1932.

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Fluorescent Probes for Molecular Imaging

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Alexa
Fluor 488 C5 maleimide, Alexa Fluor
647 C2 maleimide, Alexa Fluor 405 NHS ester, and Alexa Fluor 647 NHS
ester were purchased from Molecular Probes. Heparin (low molecular
weight Heparin) was obtained from Fisher Scientific UK. Ammonium acetate,
thioflavin T (ThT), and dithiothreitol (DTT) were purchased from Sigma.
pFTAA was a kind gift from Therese Klingstedt.

Kundel F., De S., Flagmeier P., Horrocks M.H., Kjaergaard M., Shammas S.L., Jackson S.E., Dobson C.M, & Klenerman D. (2018). Hsp70 Inhibits the Nucleation and Elongation of Tau and Sequesters Tau Aggregates with High Affinity. ACS Chemical Biology, 13(3), 636-646.

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Cardiac and Brain Imaging in Zebrafish

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Larvae were immobilized with tricaine and placed in an agarose injection mold with their hearts facing upwards. 2.3 nl of Alexa Fluor 405 NHS Ester (Thermo Fisher: A30000) or Alexa Fluor 647 10 kDa Dextran (Thermo Fisher: D22914) fluorescently conjugated tracers (10 mg/ml) were injected into the cardiac sac using Nanoject II (Drummond Scientific, Broomall, PA). Larvae were then mounted with 1.5% low gelling agarose (Sigma: A9414) in embryo water on 0.17 mm coverslips and imaged live within 2 hr post-injection. For developmental electron microscopy experiments, 2.3 nl of 5 nm NHS-activated gold nanoparticles (Cytodiagnostics: CGN5K-5–1, ~1.1¹⁴ particles/ml in PBS) were injected into the cardiac sac just as for the fluorescently conjugated tracers. After 5 min of circulation, the fish were fixed for electron microscopy. The brains of transgenic DBP-EGFP adults were fixed in 4% paraformaldehyde in PBS overnight at 4°C. Following fixation the brains were washed three times and sagittally sectioned with a cryostat (30 µm) to reveal DBP-EGFP localization within the brain.

O'Brown N.M., Megason S.G, & Gu C. (2019). Suppression of transcytosis regulates zebrafish blood-brain barrier function. eLife, 8, e47326.

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Quantifying Cell Cytoskeletal and Signaling Dynamics

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Chemical reagents used were as follows: Alexa Fluor 647 phalloidin (Cat# A22287; Thermo Fisher), Alexa Fluor plus 405 phalloidin (Cat# A30104; Thermo Fisher), D-erythro-sphingosine-1-phosphate (Cat# 860492; Avanti Polar Lipids), normal donkey serum (Cat# 017–000-121; Jackson Immunoresearch), rapamycin (Cat# R5000; LC Laboratories), puromycin dihydrochloride (Cat# P-600-100; Gold Biotechnology), insulin (Cat# I-1882; Sigma-Aldrich), human holo transferrin (Cat# T-4132; Sigma-Aldrich), sodium selenite (Cat# 5–5261; Sigma-Aldrich), FA-free BSA (Cat# A8806; Sigma-Aldrich), oleic acid (Cat# O7501; Sigma-Aldrich), collagen I, bovine (Cat# A10644-01; GIBCO BRL), Alexa Fluor 405 NHS Ester (succinimidyl ester; Cat# A30000; Thermo Fisher), Oregon Green-488–conjugated gelatin (Cat# G13186; Thermo Fisher), ascorbic acid (Cat# A4034; Sigma-Aldrich), and basic fibroblast growth factor (Cat# 234-FSE; R&D Systems), VEGF165, human (Cat# 1150–05-10; Goldbiotech), GM6001 MMP Inhibitor (Cat# CC1000; Sigma-Aldrich), MT1-MMP inhibitor, NSC405020 (Cat# 444295; Sigma-Aldrich), blebbistatin (Cat# 2406–1; BioVision), and saracatinib (Cat# sc-45364607; Santa Cruz).

Collins K.B., Kang H., Matsche J., Klomp J.E., Rehman J., Malik A.B, & Karginov A.V. (2019). Septin2 mediates podosome maturation and endothelial cell invasion associated with angiogenesis. The Journal of Cell Biology, 219(2), e201903023.

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Antibody Panel for Microscopy Studies

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All the primers used in this study were purchased from Thermo Fisher, gene blocks were purchased from IDT DNA, and restriction enzymes were purchased from New England BioLabs. The primary antibodies used in this study are the following: mouse α-V5 (clone SV5-Pk2, Bio-Rad); rabbit α-V5 (ICL, RV5-45A-Z); rat α-hemagglutinin (HA, clone 3F10, Sigma); mouse α-tubulin (clone B-5-1-2, Sigma); rabbit α-Histone H3 (ab1971, Abcam); mouse α-Centrin (CrCen clone 20H5, EMD Millipore); and rabbit α-PfGAP45 [a gift from Julian Rayner at the University of Cambridge (54 (link))]. Secondary antibodies and other reagents (Alexa Fluor 405 NHS-Ester, SYTOX Deep Red Nucleic Acid Stain, and Hoechst 33342 Solution) used for microscopy were purchased from Thermo Fisher.

Gurung P., McGee J.P, & Dvorin J.D. (2024). PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum. mBio, 15(5), e02850-23.

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Antibody Reagents for Microscopy Experiments

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All the primers used in this study were purchased from Thermo Fisher, gene blocks were purchased from IDT DNA, and restriction enzymes were purchased from New England Biolabs. The primary antibodies used in this study are the following: mouse α-V5 (clone SV5-Pk2, BioRad); rabbit α-V5 (ICL, RV5-45A-Z), rat α-hemagglutinin (HA, clone 3F10, Sigma); mouse α-tubulin (clone B-5-1-2, Sigma); rabbit α-Histone H3 (ab1971, Abcam); mouse α-Centrin (CrCen clone 20H5, EMD Millipore); and rabbit α-PfGAP45 (gift from Julian Rayner at the University of Cambridge [53 (link)]). Secondary antibodies and other reagents (Alexa Fluor 405 NHS-Ester, SYTOX Deep Red Nucleic acid stain, and Hoechst 33342 solution) used for microscopy were purchased from Thermo Fisher.

Gurung P., McGee J.P, & Dvorin J.D. (2024). PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum. bioRxiv.

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Alexa Fluor-405 Antibody Conjugation

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Antibodies were conjugated to Alexa Fluor-405 molecules using a SAIVI Antibody Labeling Kit according to the manufacturers’ directions (ThermoFisher Scientific) but using Alexa Fluor-405 NHS ester (ThermoFisher Scientific), which was mixed with each antibody at an 8:1 molar ratio for 1 hr at room temperature. The reaction was then purified over the SAIVI column, with fractions collected to determine the location of the conjugate. Resulting PfCSP mAb conjugates had degree of labeling (DOL) ratios between 1.4–2.2. For determination of concentration and DOL of Alexa Fluor-405 conjugates: absorbance correction factor = 0.7; extinction coefficient (e) = 34,500. Prior to use, conjugate binding was compared to unlabeled mAbs by rPfCSP ELISA and/or Pb-PfCSP-SPZ flow cytometry to confirm binding was not dramatically altered.

Wang L.T., Pereira L.S., Flores-Garcia Y., O’Connor J., Flynn B.J., Schön A., Hurlburt N.K., Dillon M., Yang A.S., Fabra-García A., Idris A.H., Mayer B.T., Gerber M.W., Gottardo R., Mason R.D., Cavett N., Ballard R.B., Kisalu N.K., Molina-Cruz A., Nelson J., Vistein R., Barillas-Mury C., Amino R., Baker D., King N.P., Sauerwein R.W., Pancera M., Cockburn I.A., Zavala F., Francica J.R, & Seder R.A. (2020). A potent anti-malarial human monoclonal antibody targets circumsporozoite protein minor repeats and neutralizes sporozoites in the liver. Immunity, 53(4), 733-744.e8.

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Fluorescent Protein Labeling Protocol

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Protein labeling was performed as previously reported (Feric et al., 2016 (link); Ferrolino et al., 2018 (link)). NPM1 protein was labeled with Alexa Fluor 488 NHS Ester (no. A20000; Invitrogen), DCAF13-Sof/Sof-RKA/Nter proteins were labeled with Alexa Fluor 405 NHS Ester (no. A30000; Invitrogen), and UTP23 was labeled with Alexa Fluor 647 NHS Ester (no. A37573; Invitrogen) according to the manufacturer’s protocol. Briefly, labeling dye was introduced into a 50 μM protein solution prepared by dilution with storage buffer (20 mM HEPES, 500 mM NaCl, 1 mM DTT, pH = 7.5) at a concentration ratio of 1:1 and the solution was incubated for 1 h in the dark. Excessive dye was eluted by Amicon Ultra-3K centrifugal filter devices (no. UFC500396; Millipore). In addition, fluorescently labeled NPM1-A488 was mixed with unlabeled NPM1 at a 1:9 ratio in 6 M guanidine-HCl solution and dialyzed with Slide-A-Lyzer Dialysis Cassettes (3.5 K MWCO, no. 66330; Thermo Fisher Scientific) in storage buffer (20 mM HEPES, 500 mM NaCl, 1 mM DTT, pH = 7.5), followed by concentration to 300 μM by Ultra-3K centrifugal filter devices.

Zhou L., Wang S., Hu W., Liu X., Xu L., Tong B., Zhang T., Xue Z., Guo Y., Zhao J., Lu L., Fan H., Qian W., Chen J., Chen W, & Wang L. (2023). T cell proliferation requires ribosomal maturation in nucleolar condensates dependent on DCAF13. The Journal of Cell Biology, 222(10), e202201096.

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Isolation and Labeling of Photoreceptor Outer Segments

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Photoreceptor outer segments (POSs) were isolated from fresh porcine eyes (Sierra for Medical Science, http://www.sierra-medical.com) as previously described (60 (link)) and conjugated with Alexa Fluor 405 NHS Ester (Invitrogen, Thermo Fisher Scientific, A30100) as described in the product manual. fPOS preparations were quantified using Bradford assays. fPOS aliquots were stored at –80°C. Cells were treated with 10 μg/cm² fPOS and incubated at 37°C for 5 hours. All media were removed, and cells were rinsed with Dulbecco’s PBS (–/–) and then treated with trypsin for 5 minutes at 37°C. Cells were dissociated in trypsin and then diluted 1:1 in FACS buffer (1× PBS [–/–], 2.5 mM EDTA, 25 mM HEPES, pH 7.0, 1% heat-inactivated FBS, 1:2,000 DRAQ5). Samples were immediately analyzed on a ZE5 YETI (Bio-Rad). Cells were gated according to DRAQ5 labeling, and 40,000 events were collected per sample. Data were processed using FlowJo software (BD Biosciences).

Eade K.T., Ansell B.R., Giles S., Fallon R., Harkins-Perry S., Nagasaki T., Tzaridis S., Wallace M., Mills E.A., Farashi S., Johnson A., Sauer L., Hart B., Diaz-Rubio M.E., Bahlo M., Metallo C., Allikmets R., Gantner M.L., Bernstein P.S, & Friedlander M. (2023). iPSC–derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function. The Journal of Clinical Investigation, 133(9), e163771.

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