Disuccinimidyl suberate dss

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Disuccinimidyl suberate (DSS) is a chemical crosslinking agent used in biochemical and molecular biology applications. It is a homobifunctional N-hydroxysuccinimide (NHS) ester that can covalently link primary amine groups on proteins or other biomolecules. DSS has a spacer arm length of 11.4 Å, allowing it to connect target molecules.

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Lab products found in correlation

Edta free protease inhibitor, by Selleck Chemicals (2 mentions) Triton x 100, by Merck Group (2 mentions) Amicon ultra 100 k cutoff spin concentrators, by Merck Group (2 mentions) Superdex 200 10 300 gl, by GE Healthcare (2 mentions) Nupage lds sample buffer 4x, by Thermo Fisher Scientific (2 mentions) Deuterium oxide, by Eurisotop (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Divinylbenzene, by Merck Group (1 mentions) D glucose monohydrate, by Merck Group (1 mentions) Toluene, by Merck Group (1 mentions) Sodium chloride (nacl), by AppliChem (1 mentions) Acetic acid, by Carl Roth (1 mentions) Styrene, by Merck Group (1 mentions) Phosphatase inhibitor, by Selleck Chemicals (1 mentions) Sds loading buffer, by Cell Signaling Technology (1 mentions) Protein inhibitor cocktail, by Merck Group (1 mentions) Anti hace2 antibody, by R&D Systems (1 mentions) Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Sephadex g 100, by GE Healthcare (1 mentions) Bio gel p 4, by Bio-Rad (1 mentions)

Close spelling variants of this product

Disuccinimidyl suberate (DSS) (21655) Disuccinimidyl suberate (DSS) crosslinker Disuccinimidyl suberate

30 protocols using disuccinimidyl suberate dss

Deuterated Biomolecule Preparation for SANS

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Bovine serum albumin (BSA),
disuccinimidyl glutarate (DSG), urea, sodium chloride (NaCl), phosphate-buffered
saline (PBS, powder, pH 7.4, for preparing 1 L solutions), and dimethyl
sulfoxide were obtained from Sigma-Aldrich. Disuccinimidyl suberate
(DSS) was purchased from Acros Organics. Deionized water was obtained
from a Thermo Scientific apparatus (Barnstead TII Pure Water System).
Deuterium oxide (D₂O, 99.9% D), deuterated urea (D4, 98%
D), and deuterated dimethyl sulfoxide (d₆-DMSO, 99.8% D) were obtained from Eurisotop and used without further
purification. Deuterated PBS (d-PBS) powder was prepared
from hydrogenated PBS powder by exchanging the hydrogen atoms for
deuterium, dissolving the salts in D₂O, followed by freeze-drying.
Similarly, the BSA for SANS was dissolved in D₂O to remove
exchangeable protons, and after 1 day of incubation, the solution
was lyophilized. This process was performed twice.

Malo de Molina P., Le T.P., Iturrospe A., Gasser U., Arbe A., Colmenero J, & Pomposo J.A. (2022). Neat Protein Single-Chain Nanoparticles from Partially Denatured BSA. ACS Omega, 7(46), 42163-42169.

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Functionalized Polymeric Nanoparticles Synthesis

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Styrene, divinylbenzene (DVB), dimethyl sulfoxide (DMSO), (3-aminopropyl)triethoxysilane (APTES), trimethoxyvinylsilane (TMVS) and D-glucose monohydrate were purchased from Merck KGaA, toluene, 2,2 0 -azoisobutyronitrile (AIBN), 11-mercapto-1-undecanol and yeast extract from Sigma Aldrich, proteose peptone and ethanol from VWR Chemicals, sodium dodecyl sulfate (SDS) from Fluka Chemie AG, disuccinimidyl suberate (DSS) from Acros Organics, acetic acid from Carl Roth GmbH + Co. KG and NaCl from AppliChem GmbH. All chemicals were used as received.

Werner M., Glück M.S., Bräuer B., Bismarck A, & Lieberzeit P.A. (2022). Investigations on sub-structures within cavities of surface imprinted polymers using AFM and PF-QNM. Soft matter, 18(11).

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In Vivo Chemical Cross-Linking with DSS

Cited 1 time

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In vivo chemical cross-linking with disuccinimidyl suberate (DSS) (Thermo Fisher Scientific) was carried out as described [25 (link), 47 (link)] with some modifications. Cells transfected with expression plasmids for 17 h were washed three times in PBS with Ca²⁺/Mg²⁺ and then incubated with 1 mM DSS or DMSO (as control) in PBS for 1 h at room temperature. The reaction was stopped with a final concentration of 20 mM Tris (pH 7.5) for 15 min at room temperature. The samples were dissolved in NuPAGE^TM LDS sample buffer (4X) (Invitrogen) and analyzed by Western blotting.

Yu R., Liu T., Jin S.B., Ankarcrona M., Lendahl U., Nistér M, & Zhao J. (2021). MIEF1/2 orchestrate mitochondrial dynamics through direct engagement with both the fission and fusion machineries. BMC Biology, 19, 229.

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Inflammasome Activation and Cytokine Profiling

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THP-1-derived macrophages or BMDMs were seeded in 6-well plates and treated as indicated. The cell pellets were collected into 1.5 ml Eppendorf tubes and lysed in TBS buffer (50 mM Tris-HCl (GCRF, China), 150 mM NaCl (GCRF, China), 0.5% Triton X-100 (Sigma-Aldrich), PH 7.4) with phosphatase inhibitor (Roche) and EDTA-free protease inhibitor (Bimake) on a rocker for 30 min on ice, and then centrifuged at 6000 × g/4 °C for 15 min to discard the supernatants. The cell supernatants were harvested for precipitation and detected the activation of caspase-1 (p10) and the maturation of IL-1β (p17) by western blotting. The pellets were washed twice with TBS buffer and resuspend in TBS buffer containing 2 mM fresh disuccinimidyl suberate (DSS, Thermo Fisher Scientific) cross-linker at 37 °C for 30 min to crosslink with flipping the tubes every 10 min, and then spun at 6000 × g/4 °C for 15 min. The crosslinked pellets were resuspended in 25 μl 2× SDS loading buffer (Cell Signaling Technology) and boiled at 100 °C for 5 min, and analyzed by immunoblotting of anti-ASC antibody, anti-BAX antibody or anti-BAK antibody.

Wang L.Q., Liu T., Yang S., Sun L., Zhao Z.Y., Li L.Y., She Y.C., Zheng Y.Y., Ye X.Y., Bao Q., Dong G.H., Li C.W, & Cui J. (2021). Perfluoroalkyl substance pollutants activate the innate immune system through the AIM2 inflammasome. Nature Communications, 12, 2915.

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SARS-CoV-2 RBD Crosslinking and Immunoprecipitation

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HEK293TN and hACE2-HEK 293 TN cells were grown at 90% confluency in 150 mm plates. Cells were then detached in PBS. After 3 washes, cells were resuspended in PBS buffer containing 20 μg/ml of recombinant SARS-CoV-2-RBD and incubated with agitation at 4 °C for 60’. Primary amine crosslinker Disuccinimidyl Suberate (DSS) (Thermo Fisher Scientific cat no. 21555) was then added to the mix at a 2 mM final concentration. Crosslinking reactions were performed at room temperature (RT) for 30 min in agitation and were then quenched by addition of 20 mM of TRIS pH 7.4 for 15’.²⁰ (link) After two additional washes in PBS, cells were lysed in the following buffer: 50 mM TRIS pH 7.4, 150 mM NaCl, 1% TRITON and 1:100 protein inhibitor cocktail (Sigma–Aldrich cat no. P8340).
1 mg of total cell lysates were then incubated for 2 h at 4^οC in constant rotation with protein G Dynabeads (Life technologies cat. No. 10004D) previously incubated with 25 μg/ml of anti-hACE2 antibodies (R&D systems, cat no. AF933). The beads were then washed three times with lysis buffer and proteins were eluted from the beads by incubation at 95 °C for 10 min with 2X Laemmli sample buffer. Immunoprecipitated samples were then loaded on separate gels for silver staining (to control Immunoprecipitation efficiency), coomassie blue staining (for subsequent mass spectrometry analysis) and western blotting.

Miluzio A., Cuomo A., Cordiglieri C., Donnici L., Pesce E., Bombaci M., Conti M., Fasciani A., Terracciano L., Manganaro L., Toccafondi M., Scagliola A., Oliveto S., Ricciardi S., Grifantini R., De Francesco R., Abrignani S., Manfrini N, & Biffo S. (2022). Mapping of functional SARS-CoV-2 receptors in human lungs establishes differences in variant binding and SLC1A5 as a viral entry modulator of hACE2. eBioMedicine, 87, 104390.

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Inflammasome Activation and Assessment

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To induce conventional NLRP3 inflammasome activation, cells were primed with LPS (0.25 μg/mL) for 2.5 h and then treated with ATP (2.5 mM) for 30 min. In some experiments, cells were primed with LPS, washed with PBS, and then treated with ATP. To stimulate NLRC4 and AIM2 inflammasome signaling, cells were transfected with flagellin (NLRC4) using N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) or poly dA:dT (AIM2) using Lipofectamine 2000. Inflammasome activation was determined by the presence of active caspase-1 p20 and active IL-1β in culture supernatant immunoblots and extracellular IL-1β quantification using ELISA. To detect ASC oligomerization, disuccinimidyl suberate (DSS, Thermo Scientific)-mediated cross-linking assays were performed as described previously (20 (link)). To determine NLRP3 oligomerization, speck-like aggregates of NLRP3-GFP were assessed by confocal microscopy in NLRP3-GFP-expressing BMDMs.

Son S., Yoon S.H., Chae B.J., Hwang I., Shim D.W., Choe Y.H., Hyun Y.M, & Yu J.W. (2021). Neutrophils Facilitate Prolonged Inflammasome Response in the DAMP-Rich Inflammatory Milieu. Frontiers in Immunology, 12, 746032.

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Detailed Protein Conjugation Protocol

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Five-times-recrystallized HEL was a gift from QP (Tokyo, Japan). Ovalbumin (OVA) was purchased from Sigma (Mo, USA). 2,2′-Dithiodipyridine was obtained from Tokyo Kasei Kogyo (Tokyo, Japan). 1-Ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride (EDC) was purchased from Nakalai Tesque (Kyoto, Japan). N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP) and disuccinimidyl suberate (DSS) were purchased from Thermo Scientific (Rockford, USA). BioGel P4 was a product of Bio-Rad Laboratories (Richmond, CA). CM-toyopearl was obtained from Tosoh (Kyoto, Japan). Sephadex G-100 was purchased from GE Healthcare Biosciences. All other chemicals used were of the highest quality commercially available.

Ohkuri T., Yuge N., Sato K, & Ueda T. (2019). A method to induce hen egg lysozyme-specific humoral immune tolerance in mice by pre-exposition with the protein's oligomers. Biochemistry and Biophysics Reports, 20, 100679.

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Detecting BAX Oligomerization by Crosslinking

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BAX dimers and higher order oligomers were detected through chemical
crosslinking experiments (Antonsson et al.,
2001; Peng et al., 2013 (link)).
Following the indicated treatments, cells were lysed in protease
inhibitor-containing CHAPS lysis buffer on ice for 15 min. Clarified lysates
were dosed and concentrations equalized to the lysate with lowest protein
concentration using CHAPS lysis buffer. Equalized lysates were crosslinked
with fresh disuccinimidyl suberate (DSS; Thermo Fisher Scientific) dissolved
in PBS to a final concentration of 2 mM for 15 min at room temperature with
gentle, continuous inversions. Crosslinking reactions were quenched by
addition of Tris-HCl (pH 8.0) to a final concentration of 20 mM for 5 min.
Quenched crosslinking reactions were pelleted at 4ºC and
reconstituted in 2X LDS sample reducing buffer. 30% of the final product was
subjected to Western blot analysis for the detection of BAX oligomers, using
anti-BAX antibody (ABclonal). Band intensities were measured in Image Studio
Lite (LI-COR Biosciences), and data analyzed and graphed with JMP.

Wang T.S., Coppens I., Saorin A., Brady N.R, & Hamacher-Brady A. (2020). Endolysosomal Targeting of Mitochondria is Integral to BAX-mediated Mitochondrial Permeabilization During Apoptosis Signaling. Developmental cell, 53(6), 627-645.e7.

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Surface Functionalization of Biomaterial Scaffolds

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HNO₃, NaCl and NaOH were obtained from VWR International GmbH (Darmstadt, Germany). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC), N-hydroxysuccinimid (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), tetrahydrofuran (THF) and cysteamine were purchased from Sigma-Aldrich (Taufkirchen, Germany). 3,4-dihydroxybenzaldehyde (DHBA), N,N-dimethylformamide (DMF), H₂SO₄, ethylenediamine, methanol and chloroform were acquired from Merck (Darmstadt, Germany). N-(tri(hydroxymethyl)methyl)glycin (tricine) was obtained from Alfa Aesar (Karlsruhe, Germany). Disuccinimidyl suberate (DSS) and N,N′-dicyclohexylcarbodiimide (DCC) were from Thermo Fisher Scientific (Dreieich, Germany) and Interchim (Montluçon, France), respectively. The buffer ingredients KH₂PO₄, K₂HPO₄·3 H₂O, Na₂HPO₄·2 H₂O, NaHCO₃ and C₂H₃NaO₂·3 H₂O were purchased from Gruessing (Filsum, Germany). All buffers were prepared in distilled water and sterilized before use.

Kaufmann A., Hampel S., Rieger C., Kunhardt D., Schendel D., Füssel S., Schwenzer B, & Erdmann K. (2017). Systematic evaluation of oligodeoxynucleotide binding and hybridization to modified multi-walled carbon nanotubes. Journal of Nanobiotechnology, 15, 53.

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Synthesis and Characterization of Probes

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All commercial chemicals and solvents were purchased from Sigma Aldrich or Fisher Scientific and used without further purification. The identity and purity of each product was characterized by MS, HPLC, TLC, and NMR. Purity of target compounds has been determined to be >95% by LC/MS on a Waters Autopurification system with PDA, MicroMass ZQ and ELSD detector and a reversed phase column (Waters X-Bridge C18, 4.6 × 150 mm, 5 μm) eluted with water/acetonitrile gradients, containing 0.1% TFA. Stock solutions of all inhibitors were prepared in molecular biology grade DMSO (Sigma Aldrich) at 1,000× concentrations. The PU-TCO, PU-CW800 and YK5-B probes and relevant control probes, and the PU-beads and the control probes were generated using published protocols^{13 (link),19 (link),35 (link),80 (link)–85 (link)} or as described in Supplementary Notes 1. The GA-biotin probe was purchased from Sigma (SML0985). Disuccinimidyl suberate (DSS) was acquired from ThermoFisher (21655).

McNutt S.W., Roychowdhury T., Pasala C., Nguyen H.T., Thornton D.T., Sharma S., Botticelli L., Digwal C.S., Joshi S., Yang N., Panchal P., Chakrabarty S., Bay S., Markov V., Kwong C., Lisanti J., Chung S.Y., Ginsberg S.D., Yan P., DeStanchina E., Corben A., Modi S., Alpaugh M., Colombo G., Erdjument-Bromage H., Neubert T.A., Chalkley R.J., Baker P.R., Burlingame A.L., Rodina A., Chiosis G, & Chu F. (2024). Phosphorylation-Driven Epichaperome Assembly: A Critical Regulator of Cellular Adaptability and Proliferation. Research Square.

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