Key reagents and their sources were as follows: Escherichia coli LPS serotype O1101:B4 (List Biological Laboratories), Pam3CSK4 (Invivogen), murine rTNFα (Peprotech), murine rMCSF (Peprotech), nigericin (Sigma Aldrich), ATP (Sigma Aldrich), ionomycin (LC Laboratories), N-formyl-Met-Leu-Phe (Sigma Aldrich), UTP (Sigma Aldrich), H-Leu-Leu-OMe·HBr (Bachem), Lidocaine (Sigma Aldrich), R568 (Tocris), thapsigargin (LC Laboratories), disuccinimidyl suberate (Sigma Aldrich), BAPTA-AM (Molecular Probes), 2-APB (Tocris), anti-caspase-1 (P20) mouse monoclonal antibody (Casper-1) (Adipogen), Anti-ASC rabbit polyclonal antibody (N-15) and all HRP-conjugated secondary antibodies (Santa Cruz Biotechnology), murine IL-1β ELISA kit (R&D Systems), Fluo-4 AM (Life Technologies), Pluronic F-127 (Molecular Probes), probenecid (Sigma Aldrich), Propidium iodide (Life Technologies), Cytotoxicity Detection Kit-LDH (Roche). Anti-IL-1β mouse monoclonal antibody was provided by the Biological Resources Branch, National Cancer Institute, Frederick Cancer Research and Development Center (Frederick, MD).
Disuccinimidyl suberate
Manufactured by Merck Group
Sourced in United States
Disuccinimidyl suberate is a water-soluble, homobifunctional N-hydroxysuccinimide (NHS) ester crosslinking reagent. It can be used to covalently link primary amine groups in proteins, peptides, or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Nigericin, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
N formyl met leu phe, by Merck Group (1 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
Lidocaine, by Merck Group (1 mentions)
Probenecid, by Merck Group (1 mentions)
Bapta am, by Thermo Fisher Scientific (1 mentions)
Murine r mcsf, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
2 apb, by Bio-Techne (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Ionomycin, by LC Laboratories (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Cytotoxicity detection kit ldh, by Roche (1 mentions)
Anti ha, by Thermo Fisher Scientific (1 mentions)
N glycosidase f, by New England Biolabs (1 mentions)
Supersignal west femto enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Instantblue, by Abcam (1 mentions)
Anti bak antibody, by Abcam (1 mentions)
Agar powder, by Merck Group (1 mentions)
10 protocols using disuccinimidyl suberate
1
Inflammasome Activation Assay
2
Western Blot Analysis of Protein Tags
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Medium and cell lysates were subjected to SDS-PAGE. Membranes were blocked with fat-free milk (5%) with Tween (1%) for 1 hour and incubated overnight at 4°C in the presence of either a monoclonal mouse anti-V5 (Sigma-Aldrich) or anti-HA (Invitrogen) antibody. Blots were visualized using a goat antimouse horseradish peroxidase-conjugated antibody and SuperSignal West Femto enhanced chemiluminescent substrate (Thermo Scientific) according to manufacturer's instructions. Conditioned media were deglycosylated using N-glycosidase F (New England Biolabs) or covalently cross-linked with disuccinimidyl suberate (Sigma-Aldrich), according to manufacturer's instructions, and the products analyzed by Western blotting.
3
Protein-Peptide Binding by Cross-Linking
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant proteins (1 or 2 μg) were mixed with peptides in 1× phosphate-buffered saline (PBS) and incubated at RT for 30 min with gentle shaking. Chemical cross-linking was carried out with 100 μM disuccinimidyl suberate (Sigma-Aldrich) at RT for 30 min with gentle shaking. Cross-linking reactions were quenched by the addition of 50 mM tris (pH 7.5) for 5 min at RT before boiling in 1× SDS sample buffer [5 mM tris (pH 6.8), 10% glycerol, 1.6% SDS, 100 mM DTT, and 0.02% bromophenol blue] for 5 min. Protein samples were separated by SDS-PAGE, and the gels were stained with InstantBlue (Expedeon).
4
Bak Oligomerization Assay for Mitochondrial Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Bak oligomerization assay was performed as previously reported47 (link). Briefly, mitochondria were isolated and incubated with 4 mM disuccinimidyl suberate (Sigma, St. Louis, Missouri, USA) for 30 min at room temperature. Cross-linked samples were analyzed by western blotting using an anti-Bak antibody (Epitomics, Burlingame, California, USA). Rabbit anti-VDAC1 monoclonal antibodies (Abcam, Cambridge, Massachusetts, USA) were used as a mitochondrial loading control.
5
Synthesis and Characterization of Ti3AlC2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ti3AlC2 powders were purchased from Jilin 11 Technology Co., Ltd. The 1-aminopyrene (AP) and disuccinimidyl suberate (DSS) were purchased from Sigma-Aldrich Co. Ltd. The solution of hydroiodic acid (HI, 57 wt%) was obtained from Macklin Co. Ltd. All materials were used without further purification.
6
Bioconjugation Reagents and Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BSA, Dextran, fluorescein isocyanate dextran (Dextran‐FITC), 7‐(Diethylamino) coubarin‐3‐carboxylic acid N‐succinimidyl ester (7‐DCCA NHS), N‐ethyl‐N’‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC), hexamethylene diamine, disuccinimidyl suberate, agar powder, biotin‐4‐fluorescein (FITC‐biotin), Streptavidin‐horseradish peroxidase conjugate (Streptavidin‐HRP), pH 8.5 Tris‐HCl buffer, hydrogen peroxide, 10 × phosphate buffered saline (PBS), ethanol, DMSO, N,N‐dimethylformamide, and 1‐methyl‐2‐pyrrolidinone (NMP) were purchased from Sigma‐Aldrich (St. Louis, MO). FBS was purchased form Gibco (Waltham, MA). Streptavidin was purchased from Promega (Madison, WI). Alexa Fluor‐488 Tyramide was purchased from Thermo Fisher Scientific (Waltham, MA). Deionized water (18MΩ) was used in all experiments. Lambda DNA and DNA oligonucleotides were synthesized by Bioneer (Daejeon, Korea) (Table S1 , Supporting Information).
7
Bortezomib for Protein Modification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bortezomib was obtained from Millenium (Cambridge, MA, USA). Human insulin, disuccinimidyl suberate (DSS) and dithiothreitol (DTT) were from Sigma Aldrich (St. Louis, MO, USA). Glutathione reduced and oxidized assay kit was obtained from Bioassay Systems (Hayward, CA, USA).
8
Crosslinking Assay for IRE1α Oligomerization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Increasing concentrations of protein were crosslinked by incubation with 250 μM disuccinimidyl suberate (Sigma) for 45 minutes at room temperature in buffer containing 50 mM Hepes pH7.5, 120 mM NaCl, 2 mM DTT, 1 mM EDTA and 10% glycerol. The crosslinking reaction was quenched with 50 mM Tris–HCl pH 7.5. Samples were subjected to electrophoresis on a NuPAGE 4-12% Bis-Tris gel (Life technologies) and immunoblotted using an anti-IRE1α antibody (Cell Signalling Technologies).
9
Cross-linking and Immunoblot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Th0 cells (no cytokines) or differentiated Th17 cells were cross-linked with 500 µM disuccinimidyl suberate (Sigma-Aldrich) for 30 min, and then cell lysates were prepared with radioimmunoprecipitation assay buffer. The subsequent steps were performed as described in the immunoblot analysis section.
10
Heterologous Expression of Recombinant Trypsin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synthetic oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA, USA). Restriction enzymes, T4 ligase, and Q5 polymerase were purchased from New England Biolabs (Ipswich, MA, USA), and nucleoside triphosphates (dNTPs) from Jena Bioscience (Jena, Germany). The methylotrophic yeast P. pastoris strain GS115 and the Pichia expression vector (pPIC9K) were obtained from Invitrogen (Carlsbad, CA, USA). Bovine trypsin, disuccinimidyl suberate (DSS), and the chromogenic substrates benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide (Z-GPR-pNA), 4-nitrophenyl 4-guanidinobenzoate (pNPGB), and benzoyl-L-arginine-p-nitroanilide (L-BAPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-trypsin antibody (Ab-200997) and mouse anti-his tag (Ab-49936) were obtained from Abcam (Cambridge, UK). Affi-gel 10 resin was obtained from Bio-Rad laboratories (Hercules, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!