SPDP (SPDP Crosslinker)
(Synonyms: 氮-琥珀星氩氨-3(2-吡啶二硫代)-酸酯,SPDP Crosslinker) 目录号 : GC30018
SPDP (SPDP Crosslinker)是一种短链交联剂，通过NHS酯基团与吡啶二硫反应基团和半胱氨酸硫醇反应，形成可切割（可还原）的二硫键，从而促进胺和硫醇基团的偶联。
Cas No.:68181-17-9
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
一、SPDP合成白蛋白偶联物和NanoAlb-proDOX偶联物的方案[1]
1.HSA溶解在PBS-EDTA缓冲液中(20mM sodium phosphate, 150mM NaCl, 1mM EDTA, pH 7.5)，浓度为200μM。
2.在DMSO中制备SPDP原液。在PBS-EDTA缓冲液中，将50μM HSA和1mM SPDP在室温下轻摇2h，以引发反应。
3.反应结束后，通过PD-10柱除去多余的SPDP。
4.DTT溶解在醋酸钠缓冲液中(100mM sodium acetate buffer, 100mM NaCl, pH 4.5) ，终浓度23mg/ml，将DTT溶液加入到第二步反应后的溶液中，使spdp标记的HSA最终浓度约为100μM （醋酸缓冲液与PBS-EDTA缓冲液的比例保持在1:2，以保持反应体系的pH偏酸性，防止DTT还原对天然和细胞内二硫键的影响）。
5. 反应在室温下进行1h，通过PD-10柱除去多余的DTT。
6.将还原后的HSA溶液与100μM的aldoxorubicin （AlDOX，用DMSO总反应体积的20%预稀释）在PBS-EDTA缓冲液中反应，最终HSA浓度为20μM。将混合物在室温下轻轻摇晃一夜。
7.通过PD-10柱流动去除过量的AlDOX。 NanoAlb-proDOX溶液保存在PBS缓冲液中，保存在- 80℃。
二、体内SPDP交联[2]
1.培养物生长到OD600 0.8。
2.样品洗涤两次，用交联缓冲液（20mM Na-phosphate, pH 7.5 /150mM NaCl /1mM EDTA）浓缩7.5倍，在DMSO中分别用DMSO、0.2mM SPDP或0.2mM LC-SPDP孵育30min。
3.用2.5mM Tris·HCl （pH 7.4）淬火后，收获细胞并用交联缓冲液洗涤两次。
4.细胞膜溶解于8M尿素，1% Triton X-100, 20mM Na-phosphate （pH 7.5）中。
5.用50μl Ni-NTA树脂孵育1h，从溶解膜上亲和纯化配合物，首先用10mM imidazole, 8M urea, 1% Triton X-100, 20mM Na-phosphate (pH 7.0)洗涤，然后用20mM imidazole, 1% Triton X-100, 20mM Na-phosphate (pH 7.0), 0.5M NaCl, 0.1% SDS洗涤，最后用30mM imidazole, 1% Triton X-100, 20mM Na-phosphate (pH 7.5), 150mM NaCl, 20% glycerol洗涤。所有的洗涤都进行了三次。
6.用8M urea, 50mM Tris·HCl, 2% SDS, 0.4M imidazole (pH 6.8)洗脱蛋白质，用100mM DTT还原交联剂（37℃，30 min）从络合物中分离， 最后用10% SDS/PAGE分离。
References:
[1]. Chen L, Xu N, et,al. Nanoalbumin-prodrug conjugates prepared via a thiolation-and-conjugation method improve cancer chemotherapy and immune checkpoint blockade therapy by promoting CD8+ T-cell infiltration. Bioeng Transl Med. 2022 Jul 30;8(1):e10377. doi: 10.1002/btm2.10377. PMID: 36684090; PMCID: PMC9842047.
[2]. Lobedanz S, Bokma E, et,al. A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4612-7. doi: 10.1073/pnas.0610160104. Epub 2007 Mar 5. PMID: 17360572; PMCID: PMC1838649.
SPDP (SPDP Crosslinker) is a short-chain crosslinker that reacts with NHS ester groups and pyridyl disulfide reactive groups with cysteine thiols to form a cleavable (reducible) disulfide bond, thereby facilitating the coupling of amine and thiol groups. When SPDP reacts with free thiols, it releases a detectable byproduct—2-mercaptopyridine—allowing for easy tracking of the reaction by measuring the release of 2-mercaptopyridine at 343 nm. SPDP can be used for the synthesis of antibody-drug conjugates. The SPDP crosslinker is membrane-permeable and can crosslink within cells [1-6].
References:
[1]. Singh V, Mavila AK, et,al. Effect of lysine residue modification of ovine luteinizing hormone by heterobifunctional crosslinking reagent SPDP on subunit-subunit association, receptor binding and biological activity. Indian J Exp Biol. 1992 Nov;30(11):1093-100. PMID: 1284055.
[2]. Lobedanz S, Bokma E et,al. A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4612-7. doi: 10.1073/pnas.0610160104. Epub 2007 Mar 5. PMID: 17360572; PMCID: PMC1838649.
[3]. Chen L, Xu N, et,al. Nanoalbumin-prodrug conjugates prepared via a thiolation-and-conjugation method improve cancer chemotherapy and immune checkpoint blockade therapy by promoting CD8+ T-cell infiltration. Bioeng Transl Med. 2022 Jul 30;8(1):e10377. doi: 10.1002/btm2.10377. PMID: 36684090; PMCID: PMC9842047.
[4]. Karumuthil-Melethil S, Perez N, et,al. Dendritic cell-directed CTLA-4 engagement during pancreatic beta cell antigen presentation delays type 1 diabetes. J Immunol. 2010 Jun 15;184(12):6695-708. doi: 10.4049/jimmunol.0903130. Epub 2010 May 14. PMID: 20483724; PMCID: PMC2882504.
[5]. Zhang D, Guo Y, et,al. Expression of a recombinant FLT3 ligand and its emtansine conjugate as a therapeutic candidate against acute myeloid leukemia cells with FLT3 expression. Microb Cell Fact. 2021 Mar 10;20(1):67. doi: 10.1186/s12934-021-01559-6. PMID: 33691697; PMCID: PMC7948335.
[6]. Zhao H, Li L, Peng Z, et,al. Improved targeting delivery of WED-load immunoliposomes modified with SP-A mAb for the treatment of pulmonary fibrosis. Colloids Surf B Biointerfaces. 2023 Apr;224:113237. doi: 10.1016/j.colsurfb.2023.113237. Epub 2023 Mar 1. PMID: 36871414.
SPDP (SPDP Crosslinker)是一种短链交联剂，通过NHS酯基团与吡啶二硫反应基团和半胱氨酸硫醇反应，形成可切割（可还原）的二硫键，从而促进胺和硫醇基团的偶联。当SPDP与游离巯基反应时，能够释放出可检测的副产物—2-巯基吡啶，通过在343nm处测量2-巯基吡啶的释放量可以很容易地跟踪该反应。SPDP可用于合成抗体-药物偶联物。SPDP交联剂具有膜渗透性，可在细胞内交联[1-6]。
| Cas No. | 68181-17-9 | SDF | |
| 别名 | 氮-琥珀星氩氨-3(2-吡啶二硫代)-酸酯,SPDP Crosslinker | ||
| Canonical SMILES | O=C(ON1C(CCC1=O)=O)CCSSC2=NC=CC=C2 | ||
| 分子式 | C12H12N2O4S2 | 分子量 | 312.36 |
| 溶解度 | DMSO : 155 mg/mL (496.22 mM) | 储存条件 | -20°C, protect from light, stored under nitrogen,unstable in solution, ready to use. |
| 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 | 3.2014 mL | 16.0072 mL | 32.0143 mL |
| 5 mM | 0.6403 mL | 3.2014 mL | 6.4029 mL |
| 10 mM | 0.3201 mL | 1.6007 mL | 3.2014 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。
Neurotensin-SPDP-poly-L-lysine conjugate: a nonviral vector for targeted gene delivery to neural cells
Brain Res Mol Brain Res 1999 Jun 8;69(2):249-62.10366746 10.1016/s0169-328x(99)00114-x
We report herein the synthesis of a novel DNA delivery system and in vitro evidence of its ability to transfect cell lines by binding to the high-affinity neurotensin receptor and subsequent internalization of ligand-receptor complexes. The targeting vehicle consisted of neurotensin crosslinked with poly-L-lysine via N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). The SPDP-derivatives with either neurotensin or poly-L-lysine were purified by gel filtration. The conjugate resulting of the reaction of neurotensin-SPDP with HS-SPDP-poly-L-lysine was purified through Biogel A 1.5. The neurotensin-SPDP-poly-L-lysine conjugate was able to bind plasmidic DNAs (pSV2cat and pGreen Lantern-1) at optimal molar ratios of 1:5 and 1:6 (DNA: conjugate), respectively. The conjugate internalized those plasmids in the cell lines (N1E-115 and HT-29) bearing the high-affinity neurotensin receptor. Expression of the plasmid products, chloramphenicol acetyltransferase and green fluorescent protein, was observed in such cell lines. Both internalization and expression of the plasmids transferred by the neurotensin-SPDP-poly-L-lysine conjugate were prevented by neurotensin (1 microM) and SR-48692 (100 nM), a specific antagonist of the high-affinity neurotensin receptor. The neurotensin-SPDP-poly-L-lysine conjugate was unable to transfect cell lines lacking the neurotensin receptor (COS-7 and L-929). In rat brain, the high-affinity neurotensin receptor is expressed by specific neurons such as those of the nigrostriatal and mesolimbic dopaminergic systems. Therefore, the neurotensin-SPDP-poly-L-lysine conjugate could be a useful tool for gene delivery to those neuronal systems.
[Spectral changes of C-phycocyanin with different molar ratios of SPDP]
Guang Pu Xue Yu Guang Pu Fen Xi 2008 May;28(5):1115-7.18720813
Pure C-phycocyanin was prepared from Spirulina platensis using one-step anion-exchange chromatography. The C-PC obtained was with an absorption maximum at 620 nm and a fluorescence emission maximum at 640 nm when excited by 580 nm. SPDP is an excellent heterobifunctional crosslinker for thiolating amines. Different molar ratios of SPDP have remarkable influence on the absorption and fluorescence spectra of C-phycocyanin. The absorption maximum and fluorescence emission maximum both decreased and blue-shifted from 640 nm to 630 nm as the molar ratios of SPDP increased. It was found that the molar ratios of SPDP to C-phycocyanin was not more than 100 was appropriate to being conjugated with other biomolecules from the absorption and fluorescence spectra of C-phycocyanin.
Effect of lysine residue modification of ovine luteinizing hormone by heterobifunctional crosslinking reagent SPDP on subunit-subunit association, receptor binding and biological activity
Indian J Exp Biol 1992 Nov;30(11):1093-100.1284055
The increasing use of heterobifunctional crosslinking agent in the design of hormone-carrier conjugates for selective targeting or inducing immune response against the hormone has prompted us to study the effect of epsilon-NH2 group modification of oLH-subunit, their recombination, immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of oLH alpha and oLH beta subunits were modified by using SPDP. The SPDP modified oLH alpha derivatives hybridize to native OLH beta as judged by RP-HPLC analysis. The sequential modification of alpha and beta subunits led to progressive reduction in immunoreactivity and receptor binding activities. The steroidogenic potential of oLH beta.SPDP.alpha oLH recombinant was relatively comparable. The modification of six or more epsilon-NH2 groups in oLH alpha although recombine fully with native oLH beta but failed to react to anti-oLH antibody. Moreover, steroidogenic activity was also abolished. Introduction up to four SPDP groups in oLH alpha compromised immunological and biological activities but further addition of two more SPDP groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two -NH2 groups in the receptor recognition and steroidogenic potential.
Ricin A immunotoxins of IgG and Fab of anti-CALLA monoclonal antibody: effect of water soluble long-chain SPDP on conjugate yield, immunoselectivity and cytotoxicity
Arch Pharm Res 1994 Dec;17(6):452-7.10319157 10.1007/BF02979124
The water soluble long-chain crosslinker, sulfo-succinimidyl-6-[3'-(2-pyridyldithio)-propionamido]hexanoate (S-LC-SPDP) was used to prepare ricin A chain (RTA) immunotoxins constructed with whole IgG and Fab fragments of the anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody. In this study, a) S-LC-SPDP modification efficiencies of whole IgG and Fab, b) conjugation yields of the immunotoxins prepared and c) in vitro immunoreactivity and cytotoxicity of immunotoxins constructed were examined. IgG-RTA and Fab-RTA immunotoxins were prepared with 67.3% and 57.0% conjugation yields, respectively. These long spacer intermolecular linked immunotoxins were selectively immunoreactive and cytotoxic against to immunogenic Daudi cells but little or no-binding and cytotoxic against to antigen K562 cells. Both IgG-RTA and Fab-RTA immunotoxins were 210- and 45-fold more active than intact RTA in vitro, respectively.
Conformation-specific crosslinking of mitochondrial complex I
FEBS Lett 2013 Apr 2;587(7):867-72.23454639 10.1016/j.febslet.2013.02.039
Complex I is the only component of the eukaryotic respiratory chain of which no high-resolution structure is yet available. A notable feature of mitochondrial complex I is the so-called active/de-active conformational transition of the idle enzyme from the active (A) to the de-active, (D) form. Using an amine- and sulfhydryl-reactive crosslinker of 6.8Å length (SPDP) we found that in the D-form of complex I the ND3 subunit crosslinked to the 39 kDa (NDUFA9) subunit. These proteins could not be crosslinked in the A-form. Most likely, both subunits are closely located in the critical junction region connecting the peripheral hydrophilic domain to the membrane arm of the enzyme where the entrance path for substrate ubiquinone is and where energy transduction takes place.