Bis sulfosuccinimidyl suberate

Manufactured by Thermo Fisher Scientific

Sourced in United States

Bis(sulfosuccinimidyl)suberate is a water-soluble, homobifunctional N-hydroxysulfosuccinimide ester crosslinking reagent. It can be used to covalently link primary amine-containing molecules.

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Lab products found in correlation

Microcon ym 100, by Merck Group (1 mentions) Dynabeads protein g, by Thermo Fisher Scientific (1 mentions) Dynabeads protein a, by Thermo Fisher Scientific (1 mentions) Talos f200c, by Thermo Fisher Scientific (1 mentions) Tecnai f20, by Thermo Fisher Scientific (1 mentions) Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions) Falcon 4 direct electron detector, by Thermo Fisher Scientific (1 mentions) Glacios, by Thermo Fisher Scientific (1 mentions) 4 to 15 polyacrylamide gel, by Bio-Rad (1 mentions) Pierce protein a g magnetic beads, by Thermo Fisher Scientific (1 mentions) Gfp trap a beads, by Proteintech (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions) Protein g sepharose, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Dulbecco s phosphate buffered saline, by Merck Group (1 mentions) 3 aminopropyltriethoxysilane, by Merck Group (1 mentions) Startingblock blocking buffer, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Glycerol, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Bis(sulfosuccinimidyl)suberate (BS3) (37 citations) Bis(sulfosuccinimidyl) suberate (BS3) cross-linker (2 citations)

12 protocols using bis sulfosuccinimidyl suberate

Hsp90-peptide/OVA conjugate preparation

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OVA was dialyzed for 36 h at 4°C with several changes of the buffer (PBS) to remove degradation products as well as possible contaminating peptides from the solution. Small peptides, PL19 (PDEVSGLEQLESIINFEKL) were loaded onto Hsp90 in solution at a molar ratio of 50:1 in PBS, incubated for 10 min at 45–50°C, and cooled at room temperature for 30–40 min. For Hsp90–OVA conjugate preparation, soluble Hsp90 and excess OVA (1:2 ratio) were mixed and incubated for 10 min at 45°C. The solution mixtures were then incubated at room temperature for 30 min. Free OVA was removed using Microcon YM-100 (Millipore, Bedford, MA) with a 100-kDa cutoff. Alexa 488- and Alexa 555-labeled Hsp90-peptide/OVA (Hsp90.PC) conjugates were made according to the manufacturer’s protocol (Invitrogen). For anti–SREC-I Ab-OVA preparation, OVA was coupled to anti–SREC-I Ab using bis-(sulfosuccinimidyl) suberate as described by the manufacturer (Thermo Fisher Scientific, Waltham, MA) and labeled with Alexa fluorochromes as described for Hsp90 and purified using Microcon YM-100 ultrafiltration (Millipore, Billerica, MA).

Murshid A., Gong J, & Calderwood S.K. (2014). Hsp90-peptide complexes stimulate antigen presentation through the class II pathway after binding scavenger receptor SREC-I. Immunobiology, 219(12), 924-931.

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Antibody-Dynabeads Conjugation Protocol

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Conjugation of the anti-OsTGAP1 antibody or normal rabbit IgG to Dynabeads Protein G (Invitrogen) was performed using bis[sulfosuccinimidyl] suberate (Thermo Fisher, MA, USA) as the cross-linking reagent according to the manufacturer’s protocol.

Miyamoto K., Matsumoto T., Okada A., Komiyama K., Chujo T., Yoshikawa H., Nojiri H., Yamane H, & Okada K. (2014). Identification of Target Genes of the bZIP Transcription Factor OsTGAP1, Whose Overexpression Causes Elicitor-Induced Hyperaccumulation of Diterpenoid Phytoalexins in Rice Cells. PLoS ONE, 9(8), e105823.

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Immunoprecipitation of LXRα from Protein Samples

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One mg protein sample was precleared with Dynabeads Protein A (Thermo Fisher, UK, Cat.100002D) and 1 μg isotype IgG2a (Cell Signalling, USA, Cat. 61656S). Immunoprecipitation was performed by incubating 40 μL of Dynabeads Protein A coupled to 2 μg of anti-IgG2a or 2μg of anti-LXRα using bis(sulfosuccinimidyl)suberate (Thermo Fisher, UK; Cat. A39266) with 1 mg protein sample overnight at 4°C. Dynabeads were washed 3 times with 10 mM Tris-HCl, 50 mM KCl (pH 7.5) and sample eluted in 20 μL of NUPAGE LDS sample loading buffer containing 100 mM DTT, then heated at 70°C for 10 min 10 at 70°C. The supernatant was transferred to a new Eppendorf tube after being separated from Dynabeads using a magnetic separator (Promega, UK, Cat. CD4002).

Lianto P., Hutchinson S.A., Moore J.B., Hughes T.A, & Thorne J.L. (2021). Characterization and prognostic value of LXR splice variants in triple-negative breast cancer. iScience, 24(10), 103212.

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Cryo-EM Imaging of Cross-Linked Protein Complex

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The freshly reconstituted protein complex was cross-linked with 1 mM bis-sulfosuccinimidyl suberate (Thermo Fisher Scientific) on ice for 30 min. The reaction was quenched by 50 mM tris (pH 8.0) for an additional 10 min. Four microliters of aliquots of samples at 0.1 to 0.15 mg/ml was applied to graphene oxide–coated Quantifoil R1.2/1.3 gold 400-mesh grids (Electron Microscopy Sciences). The grids were then blotted and vitrified in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific). Vitrified grids were screened in Talos F200C (Thermo Fisher Scientific) and Tecnai F20 (FEI) transmission electron microscopes to optimize the freezing conditions.
Cryo-EM data were collected in Glacios (Thermo Fisher Scientific) equipped with Falcon-4 direct electron detector operated at 200 kV in electron counting mode. Movies were collected at a nominal magnification of 150,000× and a pixel size of 0.92 Å in EER format. A total dose of 40 e⁻/Å² per movie was used with a dose rate of 5 to 6 e⁻/Å² per second. A total of 11,803 movies were recorded by automated data acquisition with EPU (Thermo Fisher Scientific).

Ito F., Alvarez-Cabrera A.L., Liu S., Yang H., Shiriaeva A., Zhou Z.H, & Chen X.S. (2023). Structural basis for HIV-1 antagonism of host APOBEC3G via Cullin E3 ligase. Science Advances, 9(1), eade3168.

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Immunoprecipitation of Toxoplasma Bradyzoites

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HFF were infected with PruΔku80 parasites at a multiplicity of infection (MOI) of 1. Parasites were differentiated to bradyzoites and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40). Bradyzoite lysates were precleared by incubation with protein G agarose beads for 30 min at 4°C to remove nonspecifically bound proteins. Then, precleared samples were incubated with protein G agarose beads covalently cross-linked with MAb bB6 using bis(sulfosuccinimidyl)suberate (Thermo Scientific) according to the manufacturer’s instructions. Precleared samples were incubated with MAb bB6 beads overnight at 4°C while rotating. Beads were washed twice with RIPA buffer before elution in Laemmli sample buffer. MAb bB6, lysate input, bead flowthrough, RIPA washes, and bead eluates were resolved on a 4-to-15% polyacrylamide gel (Bio-Rad) and stained with Coomassie blue. Bands in the eluate lanes were excised and analyzed by mass spectrometry.

Tomita T., Mukhopadhyay D., Han B., Yakubu R., Tu V., Mayoral J., Sugi T., Ma Y., Saeij J.P, & Weiss L.M. (2021). Toxoplasma gondii Matrix Antigen 1 Is a Secreted Immunomodulatory Effector. mBio, 12(3), e00603-21.

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Cell Lysis and Protein Cross-linking

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Cells were lysed in 0.5% Triton X-100 buffer (20 mM Tris HCl, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose, and 0.5% Triton X-100) for 20 min. Lysates were then centrifuged at 5000× g for 10 min. The insoluble pellets were reacted with 2 mM bis(sulfosuccinimidyl)suberate (Thermo Fisher Scientific) for 30 min and the reactions were terminated by an excess amount of glycine.

Karasawa T., Komada T., Yamada N., Aizawa E., Mizushina Y., Watanabe S., Baatarjav C., Matsumura T, & Takahashi M. (2022). Cryo-sensitive aggregation triggers NLRP3 inflammasome assembly in cryopyrin-associated periodic syndrome. eLife, 11, e75166.

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PARP1 Activation and DNA Binding Assays

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All primers for making mutations and all DNA oligomers for activating PARP1 were purchased from IDT. The double stranded DNA (p18mer) used as a control DNA has the sequence: 5’-phosphate-GGGTTGCGGCCGCTTGGG-3’. The p18mer with single stranded telomeric sequences added GTGTGGGTGTG to both 3’-ends of p18mer. The sequence of the double stranded 26mer labeled with Alexa488 on the 5’-end and Alexa546 on the other 5’-end was 5’-GCCTACCGGTTCGCGAACCGGTAGGC-3’. For the experiment with Cdc13, the dsDNA sequence of GGGTTGCGGCCGCTTGCG was used with the added 3’-overhangs of GTGTGGGTGTG. The DNA fragments 165mer and 621mer as well as the intact plasmid of 4599 bp were prepared as described (Dyer et al., 2004 (link); Winkler et al., 2011 (link)). All restriction enzymes were purchased from New England BioLabs. NAD⁺ was purchased from Sigma-Aldrich. Bis(sulfosuccinimidyl)suberate was purchased from ThermoFisher. Cdc13 protein was a kind gift from the laboratory of Dr. Deborah Wuttke (University of Colorado, Boulder). All other reagent and equipment sources are listed in the methods below.

Rudolph J., Muthurajan U.M., Palacio M., Mahadevan J., Roberts G., Erbse A.H., Dyer P.N, & Luger K. (2021). The BRCT Domain of PARP1 Binds Intact DNA and Mediates Intrastrand Transfer. Molecular cell, 81(24), 4994-5006.e5.

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Crosslinking Antibody to Capture NCKX1

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About 300 to 400 μl of Pierce Protein A/G magnetic beads (#88802, Thermo Scientific) were equilibrated with PBS and incubated with 750 μl of 5 mM bis(sulfosuccinimidyl)suberate (#21580, Thermo Scientific) for 20 min at room temperature. bis(sulfosuccinimidyl)suberate was removed, and beads were incubated for 40 min at 22 °C with 200 μg of the PMe 2D9 antibody (or the anti-Myc mAb serving as a control) dissolved in 750 μl PBS. The cross-linking reaction was quenched with 100 mM Tris HCl, pH 8.0, followed by equilibrating the beads with PBS. To characterize the ability of cross-linked PMe 2D9 antibodies to precipitate NCKX1, 5 μg of PMe 2D9 beads (or control Myc-beads) was incubated with 100 μg of rod outer segment (ROS) membranes solubilized in PBS containing 0.5% dodecyl maltoside. The beads were washed twice with the same buffer, and bound material was eluted with 100 mM Tris HCl (pH 6.8) containing 2% SDS and 10% glycerol. Eluted proteins were analyzed by Western blotting using the PMe 2D9 antibody.

Skiba N.P., Cady M.A., Molday L., Han J.Y., Lewis T.R., Spencer W.J., Thompson W.J., Hiles S., Philp N.J., Molday R.S, & Arshavsky V.Y. (2021). TMEM67, TMEM237, and Embigin in Complex With Monocarboxylate Transporter MCT1 Are Unique Components of the Photoreceptor Outer Segment Plasma Membrane. Molecular & Cellular Proteomics : MCP, 20, 100088.

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Immunoprecipitation of APC and MINK1 Proteins

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For APC IPs, 40-μL protein G-sepharose (Sigma-Aldrich, catalog no. P-3296) was washed with protein lysis buffer and incubated for 12 hours with 80 μg (for AE-MS)/20–40 μg (for Western blotting) of ALI-12–28/C-APC 41.1 antibody (both CRUK) or control V5 tag antibody (kind gift of Ron Hay) at 4°C on a rotating wheel. Antibodies were cross-linked to sepharose using bis[sulfosuccinimidyl]suberate (Thermo Fisher Scientific, catalog no. 21580). Antibody-crosslinked sepharose was incubated with pooled cell lysates harvested from 5 15-cm dishes [(AE-MS and validation co-immunoprecipitation (co-IPs)]/10-mg protein lysate (all other APC co-IPs) for 12 hours at 4°C on a rotating wheel.
For GFP IPs, 15-μL GFP-Trap_A Beads (Chromotek, catalog no. gta-100) were washed twice with PBS and twice with protein lysis buffer. Lysates harvested from one 15-cm dish of U2OS Flp-In T-Rex MINK1-GFP/GFP-MINK1/GFP cells grown for 2 days in media containing 75 ng/mL Tetracycline was incubated with the beads for 4 hours at 4°C on a rotating wheel.
Beads were washed repeatedly with 20 mmol/L Tris-HCl ph 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.05% Triton X-100, and 5% glycerol (for APC IPs), or protein lysis buffer (for GFP-IPs). Proteins were eluted by boiling with 1.3x NuPAGE LDS Sample Buffer (Thermo Fisher Scientific, catalog no. NP0008).

Popow O., Paulo J.A., Tatham M.H., Volk M.S., Rojas-Fernandez A., Loyer N., Newton I.P., Januschke J., Haigis K.M, & Näthke I. (2019). Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and β-Catenin-Independent Targets by Proteomics. Molecular cancer research : MCR, 17(9), 1828-1841.

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Immunoprecipitation of BKα-γ1 Complex

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The BKα–γ1 complex was solubilized from HEK-293 cells with 1% n-dodecyl-β-d-maltoside in TBS buffer (50 mM Tris and 150 mM NaCl, pH 7.6). After centrifugation at 17,000 g for 10 min, the solubilized BKα–γ1 complex in the supernatant was incubated with immobilized mouse monoclonal anti-BKα antibody (L6/60; NeuroMab) for 2 h, and the immunoprecipitated channel complex was washed three times (10 min each time) and then eluted from beads with Laemmli sample buffer. Protease inhibitor cocktail (Roche) was used throughout the procedure. Immobilization of antibody was achieved by covalently cross-linking to protein-A/G agarose beads with bis(sulfosuccinimidyl)suberate (Thermo Fisher Scientific) in a procedure following the manufacturer’s instruction. The eluted proteins were separated on 40–20% gradient SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed with mouse monoclonal anti-V5 antibody to detect the V5-tagged BKα and γ1 subunits.

Li Q., Guan X., Yen K., Zhang J, & Yan J. (2016). The single transmembrane segment determines the modulatory function of the BK channel auxiliary γ subunit. The Journal of General Physiology, 147(4), 337-351.

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