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Nhs peg4 azide

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NHS-PEG4-Azide is a heterobifunctional crosslinking reagent composed of an N-hydroxysuccinimide (NHS) ester and an azide group, with a polyethylene glycol (PEG) spacer. It is used to facilitate the conjugation of biomolecules, such as proteins or peptides, to other molecules or surfaces.

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Lab products found in correlation

Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions) Ethanedithiol, by Merck Group (2 mentions) Noxa bh3 peptide, by AnaSpec (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Hochest 33342, by Thermo Fisher Scientific (1 mentions) Lncap cell line, by American Type Culture Collection (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Iodoacetamide, by Merck Group (1 mentions) Lectins, by Vector Laboratories (1 mentions) Sequencing grade modified trypsin, by Promega (1 mentions) Dbco cy3, by Merck Group (1 mentions) Febabe, by Dojindo Laboratories (1 mentions) Molecular weight cutoff centrifugal filters, by Merck Group (1 mentions) Amicon ultra centrifugal filter unit, by Merck Group (1 mentions) Centricon, by Merck Group (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)

8 protocols using nhs peg4 azide

1

Cell Line Preparation and Reagent Sourcing

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PNT2 and LNCaP cell lines were obtained from the American Type Culture Collection (ATCC). Lectins were purchased from Vector Laboratories. FeBABE was purchased from Dojindo Molecular Technologies. Dithiothreitol (DTT), iodoacetamide (IAA), DBCO-NH2, and DBCO-Cy3 were purchased from Sigma-Aldrich. Phosphate Buffered Saline (PBS), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, NHS-PEG4-Azide, and Hochest 33342 were purchased from ThermoFisher Scientific. Sequencing Grade Modified Trypsin was purchased from Promega.
Xie Y., Sheng Y., Li Q., Ju S., Reyes J, & Lebrilla C.B. (2020). Determination of the glycoprotein specificity of lectins on cell membranes through oxidative proteomics. Chemical Science, 11(35), 9501-9512.
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2

Chemoselective Protein Conjugation Workflow

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Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS), N-Hydroxysuccinimide (NHS), barium hydroxide, and ethanedithiol were purchased from Sigma Aldrich. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC), NHS-PEG4-Azide, and Phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4) were purchased from Thermo Fisher Scientific. The carboxylated silica beads (3 μm) were purchased from Microspheres-Nanospheres. Biotin-modified reverse primer, BsaI-restriction site included forward primer and other DNA oligonucleotides (see sequences in Supplementary Table 1) were purchased from Integrated DNA Technologies (IDT). The pET-26b (+) plasmid was received from EMD Millipore Sigma. All synthetic peptides were purchased from GenScript. Noxa BH3 peptide was purchased from Anaspec. (See sequences in Supplementary Table 2).
Shrestha P., Yang D., Tomov T.E., MacDonald J.I., Ward A., Bergal H., Krieg E., Cabi S., Luo Y., Nathwani B., Johnson-Buck A., Shih W.M, & Wong W.P. (2021). Single-Molecule Mechanical Fingerprinting with DNA Nanoswitch Calipers. Nature nanotechnology, 16(12), 1362-1370.
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3

Modification of CPMV for Drug Delivery

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CPMV was first modified with azide linkers using an overnight reaction of 6000 molar excess of N-hydroxysuccinimide (NHS)-PEG4-azide (Thermo Scientific) with a 2 mg/mL concentration of CPMV in 0.1 M potassium phosphate (KP) buffer, pH 7.0 with 10% (v/v) DMSO. Purification was performed by ultracentrifugation pelleting at 42 000 rpm. To further display carboxyl dendrons on CPMV (CPMV*), alkyne-functionalized carboxyl dendrons were attached using 2400 molar excess with 2 mg/mL CPMV-N3 in 0.1 M KP buffer, pH 7.0 in the presence of 20 mM aminoguanidine, 20 mM l-ascorbic acid, and 2 mM CuSO4 that was added together with 10 mM tris(benzyltriazolylmethyl)-amine (THPTA). 10 kDa molecular weight cutoff centrifugal filters (Millipore) were used to purify the reaction after 2 h at room temperature. Drug-labeled formulations were then made by incubating 1000 molar excess of PS (from 50 mg/mL stock) with CPMV* in 10 mM KP buffer, pH 7.8 overnight, then removing unattached PS by ultracentrifugation pelleting in 0.1 M KP buffer, pH 7.8, which was also the buffer used for resuspension of the particles. Any aggregates were removed by a clearing spin at 10 000 rpm for 10 min before obtaining the final solution.
Wen A.M., Lee K.L., Cao P., Pangilinan K., Carpenter B.L., Lam P., Veliz F.A., Ghiladi R.A., Advincula R.C, & Steinmetz N.F. (2016). Utilizing Viral Nanoparticle/Dendron Hybrid Conjugates in Photodynamic Therapy for Dual Delivery to Macrophages and Cancer Cells. Bioconjugate chemistry, 27(5), 1227-1235.
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4

Synthesis of Functional Dendrimer Crosslinkers

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To synthesize each functional dendrimer crosslinker, 1.54 μmol of PAMAM dendrimer G3 (Sigma), G5 (Sigma) or G7 (Sigma) was dissolved in 2 ml of methanol, respectively. To modify half of the amine branches on the surface of each dendrimer, 24.64, 98.56 or 394.24 μmol of SPB (Thermo Fisher Scientific) was added in the solution containing dendrimer G3, G5 or G7, respectively (Total available amines on the surface of each dendrimer are 32, 128 and 512 for G3, G5 and G7). To generate a site to attach bridge linker, 1.54 μmol of NHS-PEG4-Azide (Thermo Fisher Scientific) was added to each dendrimer reaction solution. Then 5 μl of Et3N (Sigma) was added to each reaction solution to catalyze the reaction. The reaction mixture was stirred overnight at room temperature before the addition of 100 μl of Ac2O (Sigma) to modify the rest unreacted amine branches with acetyl groups. Each reaction mixture was stirred for another 24 h at room temperature before the addition of 3 ml of water to neutralize the reaction. Each dendrimer solution was purified and concentrated with a 3 KDa Amicon Ultra Centrifugal Filter Unit (Millipore) by centrifugation at 14,000g for 10 min at room temperature.
You Q., Cheng A.Y., Gu X., Harada B.T., Yu M., Wu T., Ren B., Ouyang Z, & He C. (2020). Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution. Nature biotechnology, 39(2), 225-235.
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5

Bioconjugation of rhBMP2 with DNA

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Recombinant human BMP-2 (rhBMP2), obtained from Medtronic Sofamor Danek, was modified with DNA in the two-step procedure shown in Supplementary Fig. 10a. To a solution of rhBMP2 dimer (50 μM in 10 mM phosphate buffer, pH 7.5) was added NHS-(PEG)4-azide (Thermo Scientific) in DMSO to a final concentration of 200 μM (2 eq.; <1% total DMSO) and vortexed briefly. The solution was incubated at RT for 2 h, and unreacted small molecule was removed by washing the solution three times with 10 mM citrate buffer (pH 3) using an Amicon Ultra-0.5 centrifugal filter unit, 3 kDa. To the retentate was added Y*DNA-DIBAC (2 eq. in water) and sodium chloride to 100 mM and the solution was incubated at RT overnight. Unreacted DNA was removed by washing the solution three times with 10 mM citrate (pH ∼3) using an Amicon Ultra-0.5 centrifugal filter unit, 10 kDa. The ratio of DNA versus protein was calculated as 0.9:1 using UV–Vis (Supplementary Fig. 10b), subtracting the spectrum of unmodified rhBMP2, and using the extinction coefficient of Y*DNA (ε260 nm=262,100 M−1 cm−1) relative to that of rhBMP2 (ε280 nm=18,421 M−1 cm−1).
Freeman R., Stephanopoulos N., Álvarez Z., Lewis J.A., Sur S., Serrano C.M., Boekhoven J., Lee S.S, & Stupp S.I. (2017). Instructing cells with programmable peptide DNA hybrids. Nature Communications, 8, 15982.
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6

Chemoselective Protein Conjugation Workflow

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Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS), N-Hydroxysuccinimide (NHS), barium hydroxide, and ethanedithiol were purchased from Sigma Aldrich. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC), NHS-PEG4-Azide, and Phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4) were purchased from Thermo Fisher Scientific. The carboxylated silica beads (3 μm) were purchased from Microspheres-Nanospheres. Biotin-modified reverse primer, BsaI-restriction site included forward primer and other DNA oligonucleotides (see sequences in Supplementary Table 1) were purchased from Integrated DNA Technologies (IDT). The pET-26b (+) plasmid was received from EMD Millipore Sigma. All synthetic peptides were purchased from GenScript. Noxa BH3 peptide was purchased from Anaspec. (See sequences in Supplementary Table 2).
Shrestha P., Yang D., Tomov T.E., MacDonald J.I., Ward A., Bergal H., Krieg E., Cabi S., Luo Y., Nathwani B., Johnson-Buck A., Shih W.M, & Wong W.P. (2021). Single-Molecule Mechanical Fingerprinting with DNA Nanoswitch Calipers. Nature nanotechnology, 16(12), 1362-1370.
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7

PEGylation of TUT7 for Cryo-EM

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To protect the TUT7 particles from sticking to the air-water interface where they were prone to denaturation, a PEGylation modification was applied for cryo-EM sample preparation. Then 100 μl of 2 mg ml -1 TUT7 was prepared in HEPES buffer: 20 mM HEPES (pH 7.8), 100 mM NaCl and 2 mM DTT. NHS-PEG4-Azide (ThermoFisher) was added at a final concentration of 2 mM. The PEGylation reaction was performed on ice for 2 h, quenched by adding 50 mM Tris buffer (pH 8.0) followed by size-exclusion chromatography (Superdex 200 Increase 1.5/150 (GE)). Protein fractions with purity better than 95% were pooled together and concentrated with a 50 kDa cutoff Centricon (Millipore) to 1 mg ml -1 for storage at -80 °C.
Yi G., Ye M., Carrique L., El-Sagheer A., Brown T., Norbury C.J., Zhang P, & Gilbert R.J.C. (2024). Structural basis for activity switching in polymerases determining the fate of let-7 pre-miRNAs. Nature structural & molecular biology.
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8

Photocatalytic Antibody Conjugation

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The following operations were carried out at room temperature. 150 μL of a 2.0 mg/mL stock of goat α-mouse polyclonal antibody (Millipore, AP124) was combined with 15 μL of 1.0 M NaHCO3 and 10 μL of a 10 mM stock of NHS-PEG4-azide (Thermo fisher 26130) in DMSO. The mixture was incubated for 1.5 hours. After this time, the reaction mixture was passed through a ZEBA 40 kDa 2 mL desalting column which had been equilibrated with Tris-HCl (pH 8.0, 50 mM). The Tris-HCl solution (ca. 200 μL) was then treated with Osmium-alkyne (33) in DMSO (final concentration 200μM), then the components of the Click-It kit (Thermo Fisher Scientific, C10276) were added: 15 μL Cu(OAc)2, and 15 μL additive 1. The mixture was incubated for 5 minutes, then 30 μL additive 2 was added and the mixture allowed to incubate in the dark for 30 minutes. The reaction mixture was then passed through a DPBS-equilibrated desalting column to afford ca. 250 μL of Osmium-antibody stock. The photocatalyst-antibody conjugate was analyzed by BCA protein assay kit (Thermo Fisher) for total protein concentration and absorbance at 350 nm for Os catalyst concentration against serial dilutions of respective standards. The typical conjugation ratio was 1:6 (antibody:photocatalyst) using these conditions.
Tay N.E., Ryu K.A., Weber J.L., Olow A.K., Cabanero D.C., Reichman D.R., Oslund R.C., Fadeyi O.O, & Rovis T. (2022). Targeted activation in localized protein environments via deep red photoredox catalysis. Nature chemistry, 15(1), 101-109.
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