Alexafluor647 hydrazide

Manufactured by Thermo Fisher Scientific

AlexaFluor647 Hydrazide is a fluorescent dye that can be used for labeling and detection applications. It is a red-fluorescent dye with an excitation maximum at 650 nm and an emission maximum at 668 nm. The hydrazide functional group allows for conjugation to biomolecules such as proteins, carbohydrates, and nucleic acids.

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Lab products found in correlation

Qubit fluorometer, by Thermo Fisher Scientific (2 mentions) Orion star a211, by Thermo Fisher Scientific (1 mentions) Synergy water purification system, by Merck Group (1 mentions) Amicon spin filter, by Merck Group (1 mentions) Complete freund s adjuvant, by Chondrex (1 mentions) Immunization grade chick type 2 collagen, by Chondrex (1 mentions) Lactel 50 50 plga, by Durect (1 mentions) Rhodamine b rhod b, by Merck Group (1 mentions) α minimum essential medium, by Thermo Fisher Scientific (1 mentions) 1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc, by Thermo Fisher Scientific (1 mentions) Trypsin edta, by Thermo Fisher Scientific (1 mentions) Dspe peg 2000 amine, by Avanti Polar Lipids (1 mentions) 1 2 dioleoyl 3 trimethylammonium propane, by Avanti Polar Lipids (1 mentions) Sodium metaperiodate, by Merck Group (1 mentions) Bodipy fl hydrazide, by Thermo Fisher Scientific (1 mentions)

7 protocols using alexafluor647 hydrazide

Fatty Acid and Alcohol Preparation for Membrane Assays

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Fatty acids and fatty
alcohols were purchased
from Nu-Check Prep., Inc. Alexa Fluor 647 hydrazide was from Thermo
Fisher Scientific. All other chemicals were purchased from Sigma-Aldrich.
All solutions were prepared in ultrapure, deionized water (Synergy
Water Purification System, Merck) and their pH adjusted with sodium
hydroxide using an Orion Star A211 pH meter with pH and ATC probes
from Thermo Scientific (Orion 8157BNUMD). Spray solutions were prepared
by adding the desired amount of lipids to an aqueous solution of 10
mM 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and 0.2 M bicine,
pH 8.0, unless indicated otherwise. Solutions were vortexed for 2
min and tumbled overnight at 20 rpm at room temperature. Landing solutions
were prepared by adding fatty acids and fatty alcohols, at a ratio
of 2:1, to a solution of 0.2 M bicine, pH 8.0. The final lipid concentration
varied depending on the lipids used and was 0.5 mM for 9-hexadecenoic
acid/9-palmitoleyl alcohol (or palmitoleic acid/hexadecenol), 1.0
mM for 9-tetradecenoic acid/9-myristoleyl alcohol (or myristoleic
acid/tetradecenol), and 20 mM for decanoic acid/decanol, unless otherwise
stated. To ensure homogeneity, solutions were vigorously stirred for
10 min before use. Solutions containing decanoic acid and/or decanol
were initially heated to 65 °C to facilitate dissolution.

Nader S., Baccouche A., Connolly F., Abou-Ghanem M., Styler S.A., Lewis J.D., Pink D, & Mansy S.S. (2022). Model Atmospheric Aerosols Convert to Vesicles upon Entry into Aqueous Solution. ACS Earth & Space Chemistry, 7(1), 252-259.

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Purification and Labeling of Bacterial rRNA

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rRNA was purified and labeled as described previously.^{50 (link)} Briefly,
E. coli BL21 cells were harvested and homogenized, and debris was
pelleted through centrifugation. The ribosome-containing supernatant was collected, and
ribosomes were pelleted by ultracentrifugation. The ribosomes were resuspended and rRNA
was isolated using standard phenol-chloroform extraction protocols. E.
coli rRNA concentration was determined using a NanoDrop One^C and the
3′-end was labeled with Alexa Fluor 647-hydrazide (ThermoFisher) following Nelissen
et al.^{51 (link)} After labeling, rRNA was purified using isopropanol
purification and ethanol purification or using an Amicon spin filter (Millipore). An
agarose gel was used to check dye removal and sample concentrations were determined using
a NanoDrop.

Schoenmakers L.L., Yewdall N.A., Lu T., André A.A., Nelissen F.H., Spruijt E, & Huck W.T. (2023). In Vitro Transcription–Translation in an Artificial Biomolecular Condensate. ACS Synthetic Biology, 12(7), 2004-2014.

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Hyaluronic Acid-based Arthritis Treatment

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HA (250 kDa) was purchased from Creative PEGWorks and used as received. Immunization Grade Chick Type II Collagen and Complete Freund's Adjuvant were obtained from Chondrex INC. IL‐1β Rat ELISA Kit, Alexa Fluor 647 hydrazide was obtained from Life Technologies. Human RASF was obtained from BioIVT. All other chemicals are obtained from Sigma Aldrich and used without further purification.

Gao Y., Vogus D., Zhao Z., He W., Krishnan V., Kim J., Shi Y., Sarode A., Ukidve A, & Mitragotri S. (2021). Injectable hyaluronic acid hydrogels encapsulating drug nanocrystals for long‐term treatment of inflammatory arthritis. Bioengineering & Translational Medicine, 7(1), e10245.

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Lipid-based Nanoparticle Fabrication

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Lactel® 50:50 PLGA was purchased from Durect Corporation (Cupertino, CA). Fetal bovine serum (FBS), granulocyte macrophage-colony stimulating factor (GM-CSF) recombinant mouse protein, alpha minimum essential medium, trypsin/EDTA, Alexa Fluor® 647 hydrazide, and tris(triethylammonium) salt were purchased from Life Technologies Corporation (Grand Island, NY). Lipids, including 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) ((DSPE-PEG(2000)) amine), cholesterol, and 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD PE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Poly (vinyl alcohol) (PVA, MW 89,000–98,000), dichloromethane (DCM), rhodamine B (Rhod B), and BSA were purchased from Sigma-Aldrich Inc. (Saint Louis, MO). 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) was purchased from Thermo Fisher Scientific Inc. (Rockford, IL). JAWSII (ATCC® CRL-11904™) immature dendritic cells were purchased from ATCC (Manassas, VA). All other chemicals were of analytical grade.

Hu Y., Hoerle R., Ehrich M, & Zhang C. (2015). Engineering the lipid layer of lipid-PLGA hybrid nanoparticles for enhanced in vitro cellular uptake and improved stability. Acta biomaterialia, 28, 149-159.

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Fluorescent Conjugation of S-LPS

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A conjugate of oxidized S-LPS with the Alexa Fluor 647 fluorescent dye (AF-LPS) was prepared as follows. S-LPS was suspended at 1 mg/ml in 0.5 ml of 0.1 M sodium acetate (pH 5.0) and incubated with 1 mM sodium metaperiodate (Sigma-Aldrich) for 30 min on ice [33 (link)]. The reaction was stopped by adding 4 μl of glycerol and oxidized LPS was separated from other reagents by extensive, 24-h dialysis against PBS at 4°C (6–8 kDa cutoff). Fifty μl of Alexa Fluor 647 hydrazide (Invitrogen), dissolved at 50 mM in dimethyl sulfoxide, was added to 0.5 ml of LPS for 2-h incubation at room temperature. Unreacted dye was separated by dialysis. The estimated degree of labeling in this AF-LPS preparation was ~0.47:1 (AF:S-LPS).
To prepare a conjugate of native S-LPS with BODIPY FL (BO-LPS), S-LPS was suspended at 2 mg/ml in 0.25 ml of PBS, sonicated and mixed with 25 μl of 50 mM BODIPY FL hydrazide (Invitrogen) in dimethyl sulfoxide. After 40-min incubation at 37°C, the mixture was sonicated again, 0.2 ml of 0.2 M NaHCO_3, (pH 8.7) and another 25 μl of BODIPY FL hydrazide were added and the incubation was continued for further 1 h. The sample was then microfuged and the supernatant dialyzed. The estimated degree of labeling in BO-LPS was ~0.13:1.

Biedroń R., Peruń A, & Józefowski S. (2016). CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide. PLoS ONE, 11(4), e0153558.

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Quantitative Aptamer-Protein Binding Assay

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EMSA was used to verify the HiTS-RAP measured binding affinities of wild-type and mutant GFP binding aptamers to EGFP. To this end, in vitro transcribed aptamers were 3′ end-labeled with AlexaFluor647 Hydrazide (Invitrogen) as described elsewhere^{5 (link)} and quantified by Qubit Fluorometer (Invitrogen). Fluorescently labeled aptamers were mixed with recombinant GST-EGFP protein at 25°C for 45 min. The GST-EGFP concentration in the binding reaction was varied as a 2/3 dilution series starting from 500 nM, and a no protein control. The final 20 μl binding reaction was composed of 1X PBS, 5 mM MgCl₂, 0.4 U of Superase In, 1 μg of yeast tRNA, 0.005% NP-40, and 5 nM fluorescently-labeled aptamer. After addition of Bromocresol Green containing 30% glycerol to 6% final glycerol concentration, binding reactions were loaded on a 6% polyacrylamide gel (0.5X TBE, 5 mM MgCl₂) that was pre-equilibrated to 4°C and pre-run at 120V for 10 min at 4°C. Loaded gels were run at 120V for 90 min at 4°C, and then imaged with a Typhoon 9400 scanner using Cy5 settings. Images were quantified by ImageQuant5.2 software, and data were fitted to Hill Equation to determine the K_d values using Igor software. EMSA was carried out with fluorescein labeled minimal NELFapt as described elsewhere^{17 (link)}.

Tome J.M., Ozer A., Pagano J.M., Gheba D., Schroth G.P, & Lis J.T. (2014). Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling. Nature methods, 11(6), 683-688.

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Quantitative Aptamer-Protein Binding Assay

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