C1375212D、C1375212J无菌
泛素(Ub)介导的蛋白质翻译后修饰是核心调控通路，几乎参与全部细胞生理活动。除经典的介导蛋白降解功能外，现有研究证实泛素还参与细胞信号传导、DNA 损伤应答、蛋白胞内转运、细胞周期调控、炎症反应、免疫应答及细胞凋亡调控。 泛素修饰依靠异肽键完成，泛素C端与靶蛋白赖氨酸残基的ε-氨基共价结合，该过程称为泛素化修饰。泛素自身含7个赖氨酸位点(K6、K11、K27、K29、K33、K48、K63)，任意赖氨酸均可连接新的泛素分子，形成多聚泛素链。不同赖氨酸连接型泛素链赋予泛素系统多样化细胞调控功能。研究最透彻的两类泛素链为K48、K63连接型：K48多聚泛素链主要介导靶蛋白经蛋白酶体降解；K63多聚泛素链调控蛋白功能、亚细胞定位与蛋白互作。大量研究证明K11型泛素链参与细胞有丝分裂调控，未来针对该链型的研究规模将逐步比肩K48、K63。随着更多链型与疾病、特异性细胞通路的关联被报道，其余稀有泛素链的研究热度将快速上升。泛素修饰是动态可逆过程。E1激活酶、E2结合酶、E3连接酶协同催化蛋白发生单泛素化或多聚泛素化；而去泛素化酶(DUBs)可移除蛋白上单/多聚泛素链、水解泛素链。
本多聚泛素化蛋白ELISA试剂盒针对多聚泛素化蛋白复合物进行富集检测。鉴于缺乏等效的多聚泛素化蛋白绝对定量标准品，试剂盒配套细胞裂解基质参照液，参照液总蛋白经BCA法统一定值，仅用于校正实验体系偏差、统一样本上样基准，适用于同批次、同板内多组细胞样本泛素修饰水平的相对高低对比。体系的检测灵敏度极高，以总蛋白投入量计，最低阳性检出阈值为0.5 μg/mL，且在该阈值附近具备良好的浓度分辨力(可有效区分总蛋白浓度差值低至0.25 μg/mL的样本)。该特点使得本试剂盒特别适用于微量样本或低丰度原代细胞中多聚泛素化水平的动态监测，数据结果建议以"校正后OD值/总蛋白μg"的比值进行组间统计学对比。
检测原理
夹心ELISA检测体系：96孔板中包被泛素化蛋白捕获试剂，结合样本内泛素化蛋白；洗涤去除未结合杂蛋白后，加入Biotin化的检测探针；再次洗涤去除游离检测探针后，加入辣根过氧化物酶(HRP)标记链霉亲和素(SA)催化化学发光；最终加入化学发光底物，酶促发光信号强弱与样本内多聚泛素化蛋白浓度呈正相关。本试剂盒提供的标准品为某特定细胞裂解液，仅供定性或半定量筛查使用，如需对不同来源细胞样品进行精确的相对定量，建议用户进行基质效应验证。
产品组分
| C1375212 | 组件 | 物理外观 | 96 T | 储存 | 单次测试使用量 |
| C1375212A | 96孔酶标板 | 固体 | 1 EA | 室温 | 按需 |
| C1375212B | 捕获试剂 | 无色液体 | 10 μg | -20℃. | 按需 |
| C1375212C | 包被液 | 无色液体 | 20 mL | 2-8℃ | 按需 |
| C1375212D | 封闭液 | 浅黄色或黄色液体
| 40 mL | 2-8℃ | 按需 |
| C1375212E | 生物素化检测探针 | 无色液体 | 20 μL | -20℃. Store in the dark. | 1:1000稀释, 100 μL/孔 |
| C1375212F | HRP标记的链霉亲和素 | 浅棕色至棕色液体 | 10 μL | -20℃. Store in the dark. | 1:10000稀释, 100 μL/孔 |
| C1375212G | 细胞裂解基质 | 无色液体 | 200 μg | -20℃. Store in the dark. | 按需 |
| C1375212H | 10×洗涤缓冲液 | 无色液体 | 100 mL | 2-8℃ | 1:10稀释，300 μL/孔*9次 |
| C1375212I | 细胞提取缓冲液 | 无色液体 | 20 mL | 2-8℃ | 按需 |
| C1375212J | 稀释液 | 浅黄色或黄色液体 | 50 mL | 2-8℃ | 按需 |
| C1375212K | 底物试剂A | 无色液体 | 8 mL | 2-8℃ | 按需 |
| C1375212L | 底物试剂B | 无色液体 | 10 mL | 2-8℃. Store in the dark. | 按需 |
| C1375212M | 底物试剂C | 无色液体 | 2 mL | 2-8℃. Store in the dark. | 按需 |
| C1375212N | 终止液 | 无色液体 | 10 mL | 2-8℃. Store in the dark. | 50μL/孔 |
未提供需自备试剂和仪器
1. 蛋白酶抑制剂混合物(Protease Inhibitor Cocktail)以及去泛素化酶抑制剂(Deubiquitinase Inhibitors)，如PR-619,DUB抑制剂(阿拉丁P126704)、1,10-菲罗啉(阿拉丁P111141)、 N-乙基顺丁烯二酰亚胺(NEM) (阿拉丁E100553)；
2. 酶标仪：可测量双波长450 nm/620 nm，也可采用单波长450 nm；
3. 精密移液器，配有一次性吸头；
4. 洗板机(可选)；
5. 微孔板摇床；
6. 一次性手套、试剂槽
使用方法
1. 细胞提取
注意：本方案已成功应用于多种细胞系。用户应根据自身应用优化细胞提取流程。
1.1 通过离心收集细胞(非贴壁细胞)或用刮取法从培养瓶中收集贴壁细胞，使用PBS。
1.2 用冷PBS洗涤细胞两次。
1.3 移除并丢弃上清液，收集细胞沉淀。
1.4 在冰上用细胞提取缓冲液(0.5 mL)将细胞沉淀溶解30分钟，每隔10分钟涡旋一次。为了获得大致的估计，可将细胞浓度调整至约2×10⁷个细胞/毫升。使用此细胞裂解缓冲液时，细胞裂解液的蛋白浓度应为2-4 mg/mL。注：强烈建议在细胞提取缓冲液中预先加入蛋白酶抑制剂混合物(Protease Inhibitor Cocktail)以及去泛素化酶抑制剂(Deubiquitinase Inhibitors)，如PR-619 DUB抑制剂、1,10-菲罗啉、 N-乙基顺丁烯二酰亚胺(NEM)，并全程在冰上操作，以防止总蛋白降解及泛素化修饰被去泛素化酶切除，从而确保多聚泛素化蛋白复合物的完整性。
1.5 将裂解液转移至微量离心管中，在4°C下15000 rpm离心10分钟。
1.6 将澄清的提取物分装至干净的微量离心管中。这些裂解液可直接用于检测，或置于-80°C保存，避免反复冻融。
2. ELISA
可根据样本数量、复孔设置、稀释梯度按比例算试剂体积。所有试剂在检测前需恢复至室温。
2.1 配制洗涤缓冲液：将100 mL 10×洗涤缓冲液加入900 mL去离子水中，充分混合，配制成工作溶液。短期储存于4°C，长期储存于-20°C。
2.2 包被：将捕获试剂用包被液稀释至推荐浓度0.5 μg/mL，充分混匀备用。取出适量的酶标板，放入孔板架中。每孔加入100 μL配制好的包被液，确保孔底液面均匀无气泡。4℃孵育过夜。
2.3 封闭：弃去孔内包被液，使用微孔板洗板机或手动操作，向每个孔中加入300 μL洗涤缓冲液，进行1次清洗，拍干。每孔加入200 μL封闭液，37℃孵育2 h或4℃孵育过夜。
2.4 准备样本：用稀释液将所有样本稀释至同一总蛋白浓度(如100, 50, 25, 10, 5, 2, 1, 0.5, 0.25, 0.125 μg/mL)，确保上样量一致。必须设置阴性对照(如稀释液、裂解缓冲液)和阳性对照(细胞裂解基质)。所选的稀释比例应针对每个实验系统进行优化。
2.5 加样：将稀释后的样本、阴性对照和阳性对照，各取100 μL，建议做2-3个复孔，避免误差。每次检测实验都需要有阴性对照与阳性对照。37°C孵育1小时。
2.6 探针孵育：使用微孔板洗板机或手动操作，向每个孔中加入300 μL洗涤缓冲液，进行3次清洗，拍干。根据实验需要准备适量的生物素化检测探针，用稀释液按1000倍比例稀释后，向每个孔中加入100 μL稀释后的生物素化检测探针。建议用铝箔等材料覆盖酶标板避光，37°C孵育1小时。
2.7 二抗孵育：使用微孔板洗板机或手动操作，向每个孔中加入300 μL洗涤缓冲液，进行3次清洗，拍干。根据实验需要准备适量的HRP标记的链霉亲和素(SA-HRP)，用稀释液按10000倍比例稀释后，向每个孔中加入100 μL稀释后的SA-HRP。建议用铝箔等材料覆盖酶标板避光，37°C孵育1小时。
2.8 显色：使用微孔板洗板机或手动操作，向每个孔中加入300 μL洗涤缓冲液，进行3次清洗，拍干。配制显色液，显色液由底物试剂A、B、C按照4：5：1比例，现用现配。每孔加入100 μL显色液。建议用铝箔等材料覆盖酶标板避光，37°C孵育5-10min。
2.9 终止：当显色足够深时，每孔加入50 μL终止液终止反应。
2.10 读板：使用酶标仪双波长450/630 nm测定每个孔的吸光度。加终止液后，必须及时完成各孔的读数。
注意事项
1. 虽然我们建议按照上文所述的方法进行实验，但最佳实验条件会因所研究的参数不同而有所差异，必须由用户自行确定。
2. ELISA实验前请将所有组件置于室温下，使其达到室温。
3. C1375212D封闭液、C1375212J 稀释液：为无菌溶液，含营养物质，注意无菌操作，开封后请尽快使用，以防影响后续实验效果。
4. 配有可拆卸的酶标板条，可根据具体检测需求分次使用所需数量的板条进行实验。由于实验条件可能不同，每次检测时应将试剂盒中的细胞裂解基质分装一份作为校准品加入。
5. 所有液体移液操作均应使用一次性吸头和试剂槽，以避免试剂或样本之间的交叉污染。
6. 用洗涤缓冲液清洗干净，并拍干。
7. 使用最高品质的去离子水。
8. 避免接触含过氧化氢的底物溶液和酸性终止液。 终止液含硫酸，是一种强酸。操作时，请佩戴一次性手套和眼部防护用品。若接触了终止液或底物溶液，应立即用大量清水彻底冲洗皮肤，并在必要时寻求医疗帮助。
Ubiquitin (Ub)-mediated post‑translational modification is a core regulatory pathway that participates in virtually all cellular physiological activities. Beyond its canonical function in proteasomal degradation, ubiquitin is now known to be involved in signal transduction, DNA damage response, intracellular protein trafficking, cell‑cycle regulation, inflammatory responses, immune responses, and apoptosis regulation. Ubiquitin modification is achieved through an isopeptide bond: the C‑terminus of ubiquitin is covalently conjugated to the ε-amino group of a lysine residue on the target protein, a process termed ubiquitination. Ubiquitin itself contains seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), each of which can link to another ubiquitin molecule, forming polyubiquitin chains. Different lysine‑linked chain types confer diverse regulatory functions upon the ubiquitin system. The two best‑characterised polyubiquitin chain types are K48‑ and K63‑linked chains: K48‑linked chains primarily target proteins for proteasomal degradation, whereas K63‑linked chains regulate protein function, subcellular localisation, and protein-protein interactions. Substantial evidence demonstrates that K11‑linked chains are involved in mitotic regulation, and research on this chain type is expected to expand to a scale comparable to that of K48 and K63 in the future. As more associations between other chain types and diseases or specific signalling pathways are reported, interest in the less common ubiquitin chains will rapidly increase. Ubiquitination is a dynamic and reversible process. E1 activating enzymes, E2 conjugating enzymes, and E3 ligases cooperate to catalyse mono‑ or polyubiquitination, whereas deubiquitinases (DUBs) can remove mono‑/polyubiquitin moieties from proteins and hydrolyse ubiquitin chains.
This polyubiquitinated protein ELISA kit is designed for enrichment and detection of polyubiquitinated protein complexes. In the absence of an equivalent absolute quantitative standard for polyubiquitinated proteins, the kit provides a cell‑lysis matrix reference solution, the total protein of which has been determined by the BCA method. This reference is intended solely for correction of experimental system deviations and uniform sample loading, and is suitable for relative comparison of ubiquitination levels among multiple cell sample groups within the same plate and same batch. The system exhibits extremely high detection sensitivity: the minimum positive detection threshold is 0.5 μg/mL based on total protein input, and it maintains good concentration resolution near this threshold (effectively distinguishing samples with total protein concentration differences as low as 0.25 μg/mL). This feature makes the kit particularly suitable for dynamic monitoring of polyubiquitination levels in micro‑samples or low‑abundance primary cells. Data are recommended to be compared statistically between groups as "normalised OD value/μg total protein".
Detection Principle
Sandwich ELISA system: the pre‑coated 96‑well plate is immobilised with a capture reagent for ubiquitinated proteins, which binds ubiquitinated protein complexes in the sample. After washing to remove unbound contaminating proteins, a biotinylated detection probe is added. Following another wash to remove free detection probe, horseradish peroxidase (HRP)‑labelled streptavidin (SA) is added to catalyse chemiluminescence. Finally, the chemiluminescent substrate is added, and the enzymatic signal intensity is positively correlated with the concentration of polyubiquitinated proteins in the sample. The standard provided with this kit is a specific cell lysate and is intended for qualitative or semi‑quantitative screening only. For accurate relative quantification of samples from different cell sources, users are advised to perform matrix‑effect validation.
Components
| C1375212 | Components | Appearance | 96 T | Storage | Quantity Per Test |
| C1375212A | 96 well plate | Solid | 1 EA | RT | As need |
| C1375212B | Capture Reagent | Colourless liquid | 10 μg | -20℃. | As need |
| C1375212C | Coating Buffer | Colourless liquid | 20 mL | 2-8℃ | As need |
| C1375212D | Blocking Buffer | Light-yellow or yellow liquid
| 40 mL | 2-8℃ | As need |
| C1375212E | Biotinylated Detection Probe | Colourless liquid | 20 μL | -20℃. Store in the dark. | 1:1000 dilution, 100 μL/well |
| C1375212F | SA-HRP | Light brown to brown liquid | 10 μL | -20℃. Store in the dark. | 1:10000 dilution, 100 μL/well |
| C1375212G | Cell Lysis Matrix | Colourless liquid | 200 μg | -20℃. Store in the dark. | As need |
| C1375212H | 10×Wash Buffer | Colourless liquid | 100 mL | 2-8℃ | 1:10 dilution, 300 μL/well*9 times |
| C1375212I | Cell Extraction Buffer | Colourless liquid | 20 mL | 2-8℃ | As need |
| C1375212J | Dilution Buffer | Light-yellow or yellow liquid | 50 mL | 2-8℃ | As need |
| C1375212K | Substrate Reagent A | Colourless liquid | 8 mL | 2-8℃ | As need |
| C1375212L | Substrate Reagent B | Colourless liquid | 10 mL | 2-8℃. Store in the dark. | As need |
| C1375212M | Substrate Reagent C | Colourless liquid | 2 mL | 2-8℃. Store in the dark. | As need |
| C1375212N | Stop Solution | Colourless liquid | 10 mL | 2-8℃. Store in the dark. | 50 μL/well |
Components required but not supplied
1. Protease inhibitor cocktail and deubiquitinase inhibitors, e.g., PR‑619 (DUB inhibitor, Aladdin P126704), 1,10‑phenanthroline (Aladdin P111141), N‑ethylmaleimide (NEM, Aladdin E100553).
2. Microplate reader capable of dual‑wavelength measurement at 450 nm/620 nm, or single wavelength at 450 nm.
3. Precision pipettes with disposable tips.
4. Plate washer (optional).
5. Microplate shaker.
6. Disposable gloves and reagent reservoirs.
Usage Protocol
1. Cell Extraction
Note: This protocol has been successfully applied to various cell lines. Users should optimise the extraction procedure for their own applications.
1.1 Collect cells by centrifugation (non‑adherent cells) or scraping (adherent cells) from culture flasks, using PBS.
1.2 Wash cells twice with ice‑cold PBS.
1.3 Remove and discard the supernatant, and collect the cell pellet.
1.4 On ice, lyse the cell pellet in cell extraction buffer (0.5 mL) for 30 minutes, vortexing every 10 minutes. As a rough guide, adjust the cell concentration to approximately 2×10⁷ cells/mL. With this lysis buffer, the protein concentration of the lysate should be 2-4 mg/mL. Note: It is strongly recommended to supplement the cell extraction buffer with protease inhibitor cocktail and deubiquitinase inhibitors (e.g., PR‑619, 1,10‑phenanthroline, NEM) and to perform all steps on ice, to prevent total protein degradation and removal of ubiquitin modifications by deubiquitinases, thereby preserving the integrity of polyubiquitinated protein complexes.
1.5 Transfer the lysate to a microcentrifuge tube and centrifuge at 15,000 rpm for 10 minutes at 4°C.
1.6 Dispense the clarified extract into clean microcentrifuge tubes. The lysates can be used directly for the assay or stored at -80°C. Avoid repeated freeze‑thaw cycles.
2. ELISA
The volume of each reagent can be calculated proportionally based on the number of samples, replicate layout and dilution series. All reagents should be equilibrated to room temperature prior to the assay.
2.1 Preparation of Wash Buffer
Add 100 mL of 10× Wash Buffer to 900 mL of deionized water and mix thoroughly to prepare working wash solution. Store at 4 °C for short-term use, or at −20 °C for long-term storage.
2.2 Coating
Dilute capture reagent with coating buffer to the recommended concentration of 0.5 μg/mL and mix well for later use. Take an appropriate number of microplates and place them on plate holders. Add 100 μL of the prepared coating solution to each well, ensuring an even liquid layer at the well bottom without air bubbles. Incubate overnight at 4 °C.
2.3 Blocking
Discard the coating solution from wells. Add 300 μL wash buffer to each well for one wash cycle either by microplate washer or manual washing, then tap the plate dry. Add 200 μL blocking buffer to each well and incubate for 2 h at 37 °C, or overnight at 4 °C.
2.4 Sample Preparation
Dilute all samples with dilution buffer to uniform total protein concentrations (e.g., 100, 50, 25, 10, 5, 2, 1, 0.5, 0.25, 0.125 μg/mL) to maintain consistent loading volume. Negative controls (e.g., dilution buffer, lysis buffer) and positive controls (cell lysate matrix) are required. Dilution factors should be optimized individually for each experimental system.
2.5 Sample Loading
Add 100 μL of diluted samples, negative controls and positive controls to corresponding wells. Running 2–3 technical replicates is recommended to reduce experimental deviation. Both negative and positive controls must be included in every assay run. Incubate at 37 °C for 1 h.
2.6 Detection Probe Incubation
Wash each well three times with 300 μL wash buffer per well using a microplate washer or manually, then tap dry. Prepare sufficient biotinylated detection probe as required, dilute the probe 1:1000 with dilution buffer, and dispense 100 μL diluted probe into each well. Cover the microplate with aluminum foil or equivalent lightproof material to avoid light exposure, then incubate at 37 °C for 1 h.
2.7 Streptavidin Conjugate Incubation
Wash each well three times with 300 μL wash buffer per well using a microplate washer or manually, then tap dry. Prepare adequate HRP-conjugated streptavidin (SA-HRP) as needed, dilute SA-HRP 1:10000 with dilution buffer, and add 100 μL diluted SA-HRP to each well. Cover the microplate with aluminum foil or equivalent lightproof material to avoid light exposure, then incubate at 37 °C for 1 h.
2.8 Color Development
Wash each well three times with 300 μL wash buffer per well using a microplate washer or manually, then tap dry. Prepare chromogenic substrate solution right before use by mixing Substrate A, Substrate B and Substrate C at a volume ratio of 4:5:1. Add 100 μL substrate solution to each well. Shield the plate from light with aluminum foil and incubate at 37 °C for 5–10 min.
2.9 Reaction Stopping
Once adequate color development is observed, add 50 μL stop solution to each well to halt the reaction.
2.10 Plate Reading
Measure the absorbance of each well on a microplate reader using dual wavelengths at 450 nm and 630 nm. Plate reading must be carried out promptly after stop solution addition.
Precautions
1. Although we recommend the protocol described above, optimal experimental conditions may vary depending on the parameters under study and must be determined by the user.
2. Before starting the ELISA, bring all kit components to room temperature.
3. Blocking Buffer (Cat. No. C1375212D) and Dilution Buffer (Cat. No. C1375212G) are sterile solutions containing nutrients. Please pay attention to sterile operation. After opening, please use it as soon as possible to avoid affecting the subsequent experimental results.
5. Use only the microplate provided with the kit. The plate is supplied as break‑apart strips, allowing partial use. Since experimental conditions may vary, include one aliquot of the cell‑lysis matrix reference as a calibrator in each assay run.
6. Use disposable tips and reagent reservoirs for all liquid transfers to avoid cross‑contamination between reagents or samples.
7. Wash thoroughly with wash buffer and tap dry completely.
8. Use the highest‑grade deionised water.
9. Avoid contact with substrate solutions containing hydrogen peroxide and with the acidic stop solution. The stop solution contains sulfuric acid and is a strong acid. Wear disposable gloves and eye protection when handling. In case of contact with stop solution or substrate solution, rinse the affected skin immediately with plenty of water and seek medical attention if necessary.
ZJ26F0738055
危险品化学品经营许可证（带存储）