Disuccinimidyl Suberate
(Synonyms: 双琥珀酰亚胺辛二酸酯,DSS, NSC 340008) 目录号 : GC43480
Disuccinimidyl Suberate是不溶于水的同型双功能的N-羟基琥珀酰亚胺酯(NHS-酯)。Disuccinimidyl Suberate不具有带电荷的基团，所以是亲脂性和膜可渗透的，能够用于细胞内和膜内蛋白质的共轭反应。
Cas No.:68528-80-3
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
以下步骤方法是该产品的示例应用，具体应用可适当优化。
一，蛋白质交联方案
A,实验材料：
1.无水DMSO或DMF，用于溶解Disuccinimidyl Suberate；
2.反应缓冲液：使用pH为7 ~ 9的非胺类缓冲液，如100mM磷酸钠，0.15M NaCl；20mM HEPES；100mM碳酸盐/碳酸氢盐；或50mM硼酸盐。
3.淬火缓冲液：1M Tris•HCl， pH7.5 (可加入1M甘氨酸或赖氨酸)通过透析或凝胶去除未反应的实际
B,操作步骤：
1. 在反应缓冲液中制备蛋白质溶液。
2. 使用前准备交联剂。将Disuccinimidyl Suberate溶于DMSO或DMF中。称量2mg Disuccinimidyl Suberate交联剂于微量离心管中，加入216μL溶剂，使成终浓度为25mM。
3.在蛋白质样品中加入交联剂。如果蛋白质浓度大于5mg/mL，使用10倍摩尔过量的交联剂。对于样品浓度小于 5mg/mL，使用20- 50倍摩尔过量的交联剂。建议使用终浓度为0.25-5mM的交联剂。
4. 将反应混合物在室温下孵育30分钟或在冰上孵育2小时。
5. 加入淬灭缓冲液淬灭反应，终浓度为20~50mM Tris，或通过透析/脱盐除去未反应的试剂。
6. 将淬火反应在室温下孵育15分钟。
二，细胞内和细胞外交联反应
交联反应可以在悬浮细胞或培养板上的贴壁细胞上进行。在培养板上时，交联反应主要发生在暴露的细胞表面。因此，交联前，必须充分冲洗细胞表面以去除培养基，否则含胺成分会使反应猝灭。使用浓度高的细胞悬浮液交联效率较高，因为反应中只需添加较少的交联试剂。一般情况下，1-5mM的终浓度的交联试剂是有效的。随着pH值的增加，NHS反应速度加快。因此，下面的例子中pH 为8.0，以便快速完成反应。
A,所需材料：
1.交联剂溶液：在使用前，将Disuccinimidyl Suberate溶解在干燥的DMSO中，使成10-25mM。
2.磷酸盐缓冲盐水(PBS)： 20mM磷酸钠，0.15M NaCl，pH值为8。或使用pH7～9的HEPES，碳酸氢盐/碳酸盐/硼酸盐缓冲液替代。
3. 猝灭溶液:1M Tris, pH 7.5 (Tris或甘氨酸将猝灭反应。)
操作步骤：
1.将细胞悬浮在PBS (pH 8.0)中，约为25×10-6细胞/mL。
2.用冰冷的PBS (pH 8.0)洗涤细胞三次，去除含胺的培养基和蛋白质。对于细胞表面相互作用的研究，在细胞中加入配体，在4℃下孵育1小时。
3.加入Disuccinimidyl Suberate溶液至终浓度为1-5mM。
4.将反应混合物在室温下孵育30分钟。
5.加入猝灭溶液至终浓度为10-20mM Tris，淬灭反应。
6.淬灭反应在室温下进行15分钟。
注意：
1. Disuccinimidyl Suberate对湿度敏感。为了避免湿气凝结到产品上，在打开前必须将小瓶平衡到室温。
2. NHS -酯部分易水解，因此不要制备储存液，实验前现配现用。
3. 交联具有生物活性的蛋白质(如酶、抗体等)在偶联时可能导致活性损失，这可能是由分子构象变化引起的。当交联剂修饰结合底物或抗原中涉及的赖氨酸基团时，也可能发生活性损失。调整试剂与目标物的摩尔比可以改善活性损失。或者使用靶向不同官能团的交联剂。
4. NHS酯的水解是一个竞争性反应，随着ph值的增加而加快。在稀释的蛋白质或肽溶液中更容易发生水解。在浓蛋白溶液中，酰化反应更容易发生。
References:
[1].Partis M D, Griffiths D G, Roberts G C, et al. Cross-linking of protein by ω-maleimido alkanoyl N-hydroxysuccinimido esters[J]. Journal of Protein Chemistry, 1983, 2: 263-277.
Disuccinimidyl Suberate is a water-insoluble, homobifunctional N-hydroxysuccinimide ester (NHS ester), Disuccinimidyl Suberate does not possess a charged group and is lipophilic and membrane-permeable, which makes it useful for intracellular and intramembrane conjugations.
NHS esters react efficiently with primary amino groups (-NH2) in pH 7 to 9 buffers to form stable amide bonds. The reaction results in the release of N-hydroxysuccinimide. Proteins, including antibodies, generally have several primary amines in the side chain of lysine (K) residues and the N-terminus of each polypeptide that are available as targets for NHS-ester reagents. Disuccinimidyl Suberate is hydrophobic and dissolved in an organic solvent such as DMSO or DMF then added to the aqueous reaction mixture.
Application:
1. Chemical cross-linking of intracellular proteins prior to cell lysis and immunoprecipitation.
2. “Fix” protein interactions to identify weak or transient protein interactions.
3. Protein cross-linking generates bioconjugates through a single-step reaction.
4. Immobilize proteins to amine-coated surfaces.
References:
[1].Cox G W, Mathieson B J, Giardina S L, et al. Characterization of IL-2 receptor expression and function on murine macrophages[J]. Journal of immunology (Baltimore, Md.: 1950), 1990, 145(6): 1719-1726.
[2].Knoller S, Shpungin S, Pick E. The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559[J]. Journal of Biological Chemistry, 1991, 266(5): 2795-2804.
[3].Partis M D, Griffiths D G, Roberts G C, et al. Cross-linking of protein by ω-maleimido alkanoyl N-hydroxysuccinimido esters[J]. Journal of Protein Chemistry, 1983, 2: 263-277.
Disuccinimidyl Suberate是不溶于水的同型双功能的N-羟基琥珀酰亚胺酯(NHS-酯)。Disuccinimidyl Suberate不具有带电荷的基团，所以是亲脂性和膜可渗透的，能够用于细胞内和膜内蛋白质的共轭反应。
NHS-酯能够与pH7～9缓冲液中的伯氨基(-NH2)有效反应形成稳定的酰胺键，释放N-羟基琥珀酰亚胺。包括抗体在内的蛋白质通常在赖氨酸(K)残基的侧链中具有几个伯胺，并且每种多肽的N末端也可用作NHS-酯试剂的靶标。Disuccinimidyl Suberate是疏水性的，可先溶解在有机溶剂如DMSO或DMF中，然后再加入到水溶剂反应混合物中。
Disuccinimidyl Suberate 实际应用：
1.细胞裂解和免疫沉淀前细胞内蛋白的化学交联；
2.“固定”蛋白相互作用，以便识别较弱或瞬时的蛋白相互作用；
3.蛋白交联通过单步反应生成生物偶联物；
4.将蛋白固定到胺包被的表面上。
| Cas No. | 68528-80-3 | SDF | |
| 别名 | 双琥珀酰亚胺辛二酸酯,DSS, NSC 340008 | ||
| 化学名 | octanedioic acid, 1,8-bis(2,5-dioxo-1-pyrrolidinyl)ester | ||
| Canonical SMILES | O=C(CCCCCCC(=O)ON1C(=O)CCC1=O)ON1C(=O)CCC1=O | ||
| 分子式 | C16H20N2O8 | 分子量 | 368.3 |
| 溶解度 | 10mg/mL in DMSO or DMF | 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 | 2.7152 mL | 13.5759 mL | 27.1518 mL |
| 5 mM | 0.543 mL | 2.7152 mL | 5.4304 mL |
| 10 mM | 0.2715 mL | 1.3576 mL | 2.7152 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。
Cross-Linking Antibodies to Beads with Disuccinimidyl Suberate (DSS)
Cold Spring Harb Protoc 2019 Feb 1;2019(2).PMID:30710026DOI:10.1101/pdb.prot098632.
This protocol describes the cross-linking of antibodies to either Protein A or G agarose beads using Disuccinimidyl Suberate (DSS), a bifunctional cross-linker capable of directly reacting with two different amines to form stable amide bonds. Proteins, including antibodies, generally display several primary amines in the side chains of lysine (K) residues and the amino terminus of each polypeptide that represent available potential targets for N-hydroxysuccinimide (NHS)-ester cross-linking reagents. The antibody-bead cross-linking process generates a reusable resource of antibody and beads, commonly referred to as an antibody-specific resin, and can be repeatedly used for the immunoprecipitation of specific proteins if treated and stored correctly.
Disuccinimidyl Suberate cross-linked hemoglobin as a novel red blood cell substitute
Sci China C Life Sci 2005 Feb;48(1):49-60.PMID:15844357DOI:10.1360/04yc0014.
Disuccinimidyl Suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCl2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the two alpha subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pl of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.
Hemoglobin-albumin cross-linking with Disuccinimidyl Suberate (DSS) and/or glutaraldehyde for blood substitutes
Artif Cells Nanomed Biotechnol 2014 Feb;42(1):13-7.PMID:23342991DOI:10.3109/21691401.2012.762652.
Hemoglobin (Hb) derivatization for blood substitute purposes often involves multi-step processes including redox reagents such as borohydride and periodate, with possible subsequent side effects. Disuccinimidyl Suberate (DSS) allows protein cross-linking without toxic side-products, forming one-step peptide bonds with the lysine residues. Here, we report that Hb polymers were obtained using DSS, making this the first report of a single-step polymerization for blood substitutes. The increase in autooxidation rate incurred by this polymerization is completely reversed when BSA is copolymerized with Hb. Copolymerization of Hb with BSA appears to be beneficial for alleviating pro-oxidant effects, regardless of the polymerizing agent employed.
A Concise, Modular Antibody-Oligonucleotide Conjugation Strategy Based on Disuccinimidyl Ester Activation Chemistry
Chembiochem 2019 Jun 14;20(12):1599-1605.PMID:30767357DOI:10.1002/cbic.201900027.
The synthesis of antibody-oligonucleotide conjugates has enabled the development of highly sensitive bioassays for specific epitopes in the laboratory and clinic. Most synthetic schemes to generate these hybrid molecules require expensive reagents, significant quantities of input antibody, and multistep purification routes; thus limiting widespread application. Herein a facile and robust conjugation strategy is reported that involves "plug-and-play" antibody conjugation with succinimidyl-functionalized oligonucleotides, which are high yielding and compatible for use directly after buffer exchange. The succinimidyl-linked oligonucleotides are synthesized with 5'-amine-modified oligonucleotides and Disuccinimidyl Suberate (DSS), both of which are inexpensive and commercially available. Direct incubation of the resulting stable succinimidyl- oligonucleotide conjugates with commercial antibodies yields conjugates ready for use after benchtop buffer exchange. It is demonstrated that the resulting oligonucleotide-antibody and oligonucleotide-streptavidin conjugates retain potent and specific binding in activity-dependent proximity ligation imaging, and proximity ligation-mediated qPCR detection of endogenous proteins in native cellular contexts down to picogram levels of whole proteome. This DSS conjugation strategy should be widely applicable in the synthesis of protein-oligonucleotide conjugates.
Recombinant human erythropoietin (rHuEPO): cross-linking with disuccinimidyl esters and identification of the interfacing domains in EPO
Protein Sci 1993 Sep;2(9):1441-51.PMID:8401229DOI:10.1002/pro.5560020908.
Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including Disuccinimidyl Suberate or dithiobis(succinimidyl propionate). Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross-linkers, which possess an 11-12-A length of a spacer between two reacting groups. Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state. These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116. A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four alpha-helical bundles and a long stretch of beta-sheet structure are involved in the active protein.