Disuccinimidyl Glutarate
(Synonyms: 双琥珀酰亚胺戊二酸酯) 目录号 : GC43479
A homobifunctional crosslinking agent
Cas No.:79642-50-5
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >99.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cross-linking of embryos for CHIP-Seq [1]: | |
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Preparation Method |
1)After dechorionation, the embryos were weighed and transferred to a 50 ml conical tube. If there was more than 2 g of embryos, we split the sample into multiple tubes. 2) The bi-functional NHS-esters were dissolved in DMSO (Dimethyl sulfoxide). In the case of Disuccinimidyl Glutarate（DSG）, we dissolved 20 mg of DSG powder in 108 ul of DMSO to give -120 ul of a 500mM DSG stock solution. In the case of DSP, we dissolved 20 mg of powder in 85 ul of DMSO to give -99 ul of 500 mM DSP stock solution. 3) The fixation mix was prepared by adding the NHS-ester/DMSO solution to phosphate-buffered saline (PBS, 150 mM sodium chloride (NaCl) / 10 mM sodium phosphate (pH 7.6)). The optimal concentration of NHS-esters may differ between the target proteins. The maximum concentrations of Disuccinimidyl Glutarate and DSP in aqueous solution are 5 mM and 2 mM, respectively. For less than 0.5 g of embryo, prepare 5 ml of solution. Increase the volume by 5 ml for every 0.5 g of additional amount of embryo. Because the NHS-esters are unstable in aqueous solutions, this fixation mix should be prepared immediately before use. 4) Pour the fixation mix into the conical tube containing the dechorionated embryos. Add an equal volume of heptane. 5) Shake the tube vigorously for 1 h. 6) Add 550 μl of 37% formaldehyde per 5 ml of aqueous phase so that the final concentration of formaldehyde 4%. Shake vigorously for additional 15 min. 7) Centrifuge the tube in swinging bucket rotor at 500 g for one minute to pellet the fixed embryo. Remove the heptane phase by pipetting. 8) Add an equal volume of the stop solution (PBS with 125 mM Glycine and 0.1% Triton X-100) to the aqueous phase. Mix well, let stand for 2 min and then pellet the fixed embryos by centrifuging a 500 g for 1 min. 9) Remove the supernatant by pipetting. Suspend the embryos in 25 ml of stop solution and after a 2 min incubation collect the embryos by centrifugation. Spin again in the same condition. 10) Remove the supernatant, and wash the embryos by suspending in 25 ml of PBST (PBS + 0.05% Triton X-100) and spin again in the same condition. Repeat this step 2 times. 11) The fixed embryos can either be immediately processed for ChIP or transferred to a 1.5ml tube, frozen in liquid nitrogen and then stored at -80 ℃ until use. |
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Applications |
The Disuccinimidyl Glutarate -formaldehyde fixation is much more effective in capturing Elba association with Fab-7 in vivo than formaldehyde alone. The Elba1 antibody ChIP for both the 5.0 mM and 2.5 mM DSG-treated embryos showed an appreciable enrichment (6-7 fold) of Fab-7 compared with the pre-immune control. |
| Cell Crosslinking [2]: | |
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Preparation Method |
1. Remove a new vial of DSG (disuccinimidyl glutarate) from 4 ℃ storage. When it is equilibrated to room temperature, resuspend the desiccated powder in DMSO to obtain a stock solution of 0.25 M Disuccinimidyl Glutarate. Immediately before adding to the cells, prepare the final crosslinking solution of 2 mM Disuccinimidyl Glutarate in ice-cold PBS (e.g., 80 μL of 0.25 M DSG in 10 mL of PBS). Discard the unused 0.25 M Disuccinimidyl Glutarate stock solution as reconstituted Disuccinimidyl Glutarate will tend to hydrolyze and become inactive during storage. 2. Resuspend cell pellet from each sample in 10 mL of freshly prepared, ice-cold 2 mM DSG crosslinking solution. Place tubes on a roller mixer and allow crosslinking to proceed for 30 min while the solution is equilibrating to room temperature. 3. After the initial 30 min has passed, add formaldehyde to a final concentration of 1% (e.g., 270μl of a 37% stock solution in 10 mL). Incubate on a roller mixer at room temperature for an additional 10 min. 4. Immediately after 10 min has passed, quench crosslinking reaction by adding 2 M glycine to a final concentration of 0.125 M (e.g., 625 μL in 10 mL). Incubate on a roller mixer at room temperature for an additional 5 min. 5. Spin cells (300 g/5 min/4 ℃) and wash twice with ice-cold PBS. Discard solution containing formaldehyde in the appropriate waste container. Proceed immediately to lysis and sonication. |
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Applications |
Combining Disuccinimidyl Glutarate and formaldehyde crosslinking is essential to detect enrichment of TET2 on chromatin. Inclusion of the protein protein crosslinker DSG as well as titration of antibody and input chromatin resulted in improved signal-to-noise ratio. |
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References: [1]. Aoki T, Wolle D, Preger-Ben Noon E, Dai Q, Lai EC, Schedl P. Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos. Fly (Austin). 2014;8(1):43-51. doi: 10.4161/fly.26805. Epub 2013 Dec 13. PMID: 24135698; PMCID: PMC3974894. [2]. Rasmussen KD, Helin K. ChIP-Sequencing of TET Proteins. Methods Mol Biol. 2021;2272:251-262. doi: 10.1007/978-1-0716-1294-1_15. PMID: 34009619. |
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Disuccinimidyl Glutarate is an insoluble bisfunctional N-hydroxysuccinimide (NHS ester) crosslinking agent that is commonly used to pair radiolabelled ligands to cell surface receptors[1].
Combining Disuccinimidyl Glutarate and formaldehyde crosslinking is essential to detect enrichment of TET2 on chromatin. Inclusion of the protein protein crosslinker Disuccinimidyl Glutarate as well as titration of antibody and input chromatin resulted in improved signal-to-noise ratio[1]. When treated embryos with increasing concentrations of DSG (1.0 -5 mM) in the presence of an equal volume of heptane. After vigorously shaking the DSG-embryo suspension for 1 h, formaldehyde was added so that the final concentration in the water phase would be 4%. After a 15 min incubation with formaldehyde, the embryos were processed including the nuclear formaldehyde cross-linking step. The DSG-formaldehyde fixation is much more effective in capturing Elba association with Fab-7 in vivo than formaldehyde alone. The Elba1 antibody ChIP for both the 5.0 mM and 2.5 mM Disuccinimidyl Glutarate -treated embryos showed an appreciable enrichment (6-7 fold) of Fab-7 compared with the pre-immune control. The pull down of Fab-7 sequences appears to be specific as the 2 control loci, twe and Sxl, showed only a 1-1.5 fold enrichment. While there wasn t much difference between the 5.0 mM and the 2.5 mM DGS fixation, having a sufficiently high concentration of this cross-linking reagent does seem to be important, as there was only a limited enrichment with 1.0 mM Disuccinimidyl Glutarate cross-linking[2].
References:
[1]. Rasmussen KD, Helin K. ChIP-Sequencing of TET Proteins. Methods Mol Biol. 2021;2272:251-262. doi: 10.1007/978-1-0716-1294-1_15. PMID: 34009619.
[2]. Aoki T, Wolle D, et,al. Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos. Fly (Austin). 2014;8(1):43-51. doi: 10.4161/fly.26805. Epub 2013 Dec 13. PMID: 24135698; PMCID: PMC3974894.
Disuccinimidyl Glutarate 是一种不溶性双功能 N-羟基琥珀酰亚胺（NHS 酯）交联剂，通常用于将放射性标记的配体与细胞表面受体配对[1]。
结合戊二酸二琥珀酰亚胺酯和甲醛交联对于检测 TET2 在染色质上的富集至关重要。加入蛋白质交联剂戊二酸二琥珀酰亚胺酯以及滴定抗体和输入染色质可提高信噪比[1]。在等量庚烷存在的情况下，用增加浓度的 DSG (1.0 -5 mM) 处理胚胎。剧烈摇动 DSG-胚胎悬浮液 1 小时后，加入甲醛，使水相中的最终浓度为 4%。用甲醛孵育 15 分钟后，对胚胎进行处理，包括核甲醛交联步骤。 DSG-甲醛固定在体内捕获 Elba 与 Fab-7 的结合比单独使用甲醛更有效。与免疫前对照相比，5.0 mM 和 2.5 mM 戊二酸二琥珀酰亚胺酯处理的胚胎的 Elba1 抗体 ChIP 显示 Fab-7 明显富集（6-7 倍）。 Fab-7 序列的下拉似乎是特定的，因为 2 个控制基因座 twe 和 Sxl 仅显示 1-1.5 倍的富集。虽然 5.0 mM 和 2.5 mM DGS 固定之间没有太大差异，但具有足够高浓度的这种交联剂似乎很重要，因为 1.0 mM 戊二酸二琥珀酰亚胺酯交联的富集度有限[2].
| Cas No. | 79642-50-5 | SDF | |
| 别名 | 双琥珀酰亚胺戊二酸酯 | ||
| Canonical SMILES | O=C(CCCC(ON(C(CC1)=O)C1=O)=O)ON2C(CCC2=O)=O | ||
| 分子式 | C13H14N2O8 | 分子量 | 326.3 |
| 溶解度 | DMF: 10 mg/ml,DMSO: 20 mg/ml,DMSO:PBS (pH 7.2)(1:5): 0.15 mg/ml | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 3.0647 mL | 15.3233 mL | 30.6466 mL |
| 5 mM | 0.6129 mL | 3.0647 mL | 6.1293 mL |
| 10 mM | 0.3065 mL | 1.5323 mL | 3.0647 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
ChIP-Sequencing of TET Proteins
Methods Mol Biol 2021;2272:251-262.PMID:34009619DOI:10.1007/978-1-0716-1294-1_15.
TET proteins are methylcytosine dioxygenases that interact directly with chromatin to shape the DNA methylation landscape. To increase the understanding of TET protein function in a specific cellular context, it is important to be able to map the interactions between TET proteins and DNA. This ChIP-seq protocol details our procedure to analyze TET2 bound DNA in Disuccinimidyl Glutarate (DSG) and formaldehyde-crosslinked chromatin but can also be adapted to study other TET enzymes.
Capturing Chromosome Conformation Across Length Scales
J Vis Exp 2023 Jan 20;(191).PMID:36744801DOI:10.3791/64001.
Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population. A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and Disuccinimidyl Glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.
Neat Protein Single-Chain Nanoparticles from Partially Denatured BSA
ACS Omega 2022 Nov 9;7(46):42163-42169.PMID:36440132DOI:10.1021/acsomega.2c04805.
The main challenge for the preparation of protein single-chain nanoparticles (SCNPs) is the natural complexity of these macromolecules. Herein, we report the suitable conditions to produce "neat" bovine serum albumin (BSA) single-chain nanoparticles (SCNPs) from partially denatured BSA, which involves denaturation in urea and intramolecular cross-linking below the overlap concentration. We use two disuccinimide ester linkers containing three and six methylene spacer groups: Disuccinimidyl Glutarate (DSG) and disuccinimidyl suberate (DSS), respectively. Remarkably, the degree of internal cross-linking can be followed simply and efficiently via 1H NMR spectroscopy. The associated structural changes-as probed by small-angle neutron scattering (SANS)-reveal that the denatured protein has a random-like coil conformation, which progressively shrinks with the addition of DSG or DSS, thus allowing for size control of the BSA-SCNPs with radii of gyration down to 5.4 nm. The longer cross-linker exhibits slightly more efficiency in chain compaction with a somewhat stronger size reduction but similar reactivity at a given cross-linker concentration. This reliable method is applicable to a wide range of compact proteins since most proteins have appropriate reactive amino acids and denature in urea. Critically, this work paves the way to the synthesis of "neat", biodegradable protein SCNPs for a range of applications including nanomedicine.
Discovery of Cyclic Peptide Binders from Chemically Constrained Yeast Display Libraries
Methods Mol Biol 2022;2491:387-415.PMID:35482201DOI:10.1007/978-1-0716-2285-8_20.
Cyclic peptides with engineered protein-binding activity have great potential as therapeutic and diagnostic reagents owing to their favorable properties, including high affinity and selectivity. Cyclic peptide binders have generally been isolated from phage display combinatorial libraries utilizing panning based selections. As an alternative, we have developed a yeast surface display platform to identify and characterize cyclic peptide binders from genetically encoded combinatorial libraries. Through a combination of magnetic selection and fluorescence-activated cell sorting (FACS), high-affinity cyclic peptide binders can be efficiently isolated from yeast display libraries. In this platform, linear peptide precursors are expressed as yeast surface fusions. To achieve cyclization of the linear precursors, the cells are incubated with Disuccinimidyl Glutarate, which crosslinks amine groups within the displayed linear peptide sequence. Here, we detail protocols for cyclizing linear peptides expressed as yeast surface fusions. We also discuss how to synthesize a yeast display library of linear peptide precursors. Subsequently, we provide suggestions on how to utilize magnetic selections and FACS to isolate cyclic peptide binders for target proteins of interest from a peptide combinatorial library. Lastly, we detail how yeast surface displayed cyclic peptides can be used to obtain efficient estimates of binding affinity, eliminating the need for chemically synthesized peptides when performing mutant characterization.
Covalently Immobilized Regenerable Immunoaffinity Layer with Orientation-Controlled Antibodies Based on Z-Domain Autodisplay
Int J Mol Sci 2021 Dec 31;23(1):459.PMID:35008883DOI:10.3390/ijms23010459.
A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely Disuccinimidyl Glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)5), were evaluated, and DSS at a concentration of 500 μM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%.