BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光,-20°C储存,禁止冷冻。低温运输,超低温运输,禁止冷冻 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
Glysite 是一种针对IgG抗体的位点特异性偶联技术，基于Fc聚糖重塑和点击化学。该技术能够从多个物种及亚型的IgG中制备出稳定且均一的抗体偶联物。通过对抗体进行Fc N-糖链去糖基化重塑，可借助稳健的点击化学实现位点特异性偶联，所得标记程度（DOL）或药物抗体比（DAR）为2。
本试剂盒的标记流程和原理如图1所示。1.使用超滤管对待标记抗体进行溶液置换和浓缩；2.去糖基化和叠氮基团引入：Fc特异性内切糖苷酶EndoS2可将多种IgG亚型及物种的Fc糖链水解至最内侧的GlcNAc部分，利用β-1,4-半乳糖基转移酶Y289L(GalT)，将含叠氮基团的UDP-GalNAz酶促连接到暴露的GlcNAc上，生成叠氮活化的抗体；3. 使用超滤管对Azide标记抗体进行纯化和浓缩；4. 加入炔基(Alkyne)、DBCO (Dibenzocyclooctyne) 或DIBO (Dibenzylcyclooctyne)修饰的荧光蛋白（PE、 PE-Cy5、PE-Cy7、 APC等）、荧光基团(Cy3、Cy5、AF488、AF647等)、生物素(Biotin)、胺基(Amine)等标记物，通过应变促进的无铜点击化学（SPAAC）进行生物正交反应，形成稳定的三唑环连接，完成抗体标记。
每套Glysite 2位点抗体叠氮基快速标记试剂盒均包含足量试剂，可以标记约100-250μg (G1524532)、1mg (G1524533)和5mg (G1524534)的IgG抗体。试剂盒中提供的抗体浓缩管用于在抗体标记流程（见图1）的每一步对抗体进行纯化和浓缩。所有操作均在生理条件下进行，从而保持抗体的原有质量。由于偶联发生在Fc结构域，因此抗原结合位点的亲和力得以完好保留。
图1 Glysite 2位点抗体叠氮基快速标记试剂盒原理图
产品特点
产品组分
注： 发货时各组分依据储存温度(2‑8℃/-20℃)独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。 |
所需自备材料
1. 离心机。
2. 用于标记的IgG抗体（不含载体蛋白和叠氮化物）。
3. Nanodrop或分光光度计。
4. 1.5-2mL离心管。
5. 恒温水浴锅或金属浴。
重要须知
1. 试剂盒使用之前，需将所有组分恢复至室温。
2. 在开始实验前，请将试剂管进行短暂离心，以确保所有物质均聚集于管底。
3. 在整个实验方案中必须避免使用叠氮化钠。
4. 请勿重复使用Endo S2微量离心柱。由于存在前次样品残留物污染的风险，我们不建议将其用于不同抗体的处理。
5. 本试剂盒不提供Alkyne、DBCO或DIBO修饰的荧光基团等标记物。
6. 如果使用螯合剂作为标记物，则在整个操作流程中使用无金属水（痕量分析级）至关重要。抗体不得接触玻璃或金属。
7. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
8. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用说明
1 抗体缓冲液置换和浓缩
1.1 如果抗体已在50mM Tris (pH7.5) 溶液中，且抗体浓度达2-5mg/mL，且缓冲液中不含叠氮化钠，可直接进行步骤2的去糖基化反应，无须进行本步骤即抗体的缓冲液置换和浓缩。
1.2 抗体浓缩管润洗。取一支抗体浓缩管，加入0.5mL 50mM Tris (pH 7.5) ，14000 × g离心5-10 分钟，弃掉超滤内管和滤出液回收管中的液体。
注：确保浓缩管的膜面朝外，远离转子中心。
1.3 将100-250μg 待标记抗体溶液转移至浓缩管内管，用50mM Tris (pH 7.5) 将所加入的抗体稀释至500 µL刻度线处，轻轻吹打混匀。
1.4 14000 × g离心5-10 分钟，内管滞留液体积应不超过100 µL。若滞留液体积大于100µL，则重复离心，每次3分钟，直至体积减少至该范围。转移滤出液至新的离心管中，暂时保留。
注：超滤内管都有一定的漏液几率，建议超滤后，先保留滤出液回收管中溶液，确认超滤效果后再丢弃滤液，避免可能的样品损失。
1.5 向浓缩管内管加入50mM Tris (pH 7.5) 至500 μL刻度线处，轻轻吹打混匀。
1.6 重复步骤1.4和1.5两次。直至抗体体积浓缩至100 µL。
1.7 将超滤内管中的溶液转移至1.5mL离心管中。通过A280测定待标记抗体浓度，例如：A280为1.4时，蛋白浓度约为1mg/mL。
注：理论上抗体浓缩管的蛋白回收率>90%，但是部分蛋白样品回收率为60%也属于正常现象。
2 去糖基化：抗体Fc结构域上N-糖链的修饰
2.1 使用前将Endo S2 微量离心柱平衡至室温。掰断层析柱底部的封口帽，将离心柱置于微量离心管中，并拧松上盖。于 200 × g 离心 1 分钟，以去除保存液。弃去流出液。
2.2 将离心柱放回微量离心管中。向树脂上层加入 300 μL 50mM Tris (pH7.5)。于 200 × g 离心 1 分钟，弃去流出液。
2.3 重复步骤 2.2 两次（共洗涤三次）。
2.4 插回底部红色封口帽。立即向离心柱中加入抗体溶液（100 μL）。盖上上盖密封离心柱。可用Parafilm™实验室封口膜或类似材料封住管盖和底部红色封口帽。
2.5 充分悬浮树脂介质，通过颠倒混匀，并确保柱内液体可流动。在37℃下，将离心柱置于旋转混匀仪上，颠倒旋转孵育 1小时。
2.6 取下底部封口帽，将离心柱置于新的微量离心管中，并拧松上盖。于 1000 × g 离心 1 分钟，收集处理后的样品。
2.7 插回底部红色封口帽，加入 100 μL 50mM Tris (pH7.5)，盖上上盖密封。将离心柱颠倒混匀数次。
2.8 取下底部封口帽，将离心柱置于新的微量离心管中，并拧松上盖。于 1000 × g 离心 1 分钟，收集处理后的样品（即再洗脱一次）。
2.9 合并所有收集的样品（包括步骤 2.6 中的样品）。
3 抗体叠氮基团连接
3.1 按下表在1.5mL离心管中依次加入各试剂配制反应溶液，涡旋混匀，然后短暂离心管体。用Parafilm™实验室封口膜或类似材料封住管盖，然后在37°C下过夜孵育。
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4 叠氮基标记抗体的纯化和浓缩
此步骤用于去除过量的底物UDP-GalNAz。您也可以使用TBS或其他不含磷酸盐的缓冲液进行修饰抗体的纯化和收集。本试剂盒提供50mM Tris, PH7.5以供使用。
4.1 将酶促反应后的反应液转移至超滤管内管，加入50mM Tris (pH 7.5)至500 µL刻度线处，轻轻吹打混匀。
4.2 14000 × g离心5-10 分钟，内管滞留液体积应不超过100 µL。若滞留液体积大于100 µL，则重复离心，每次3分钟，直至体积减少至该范围。弃去流出液。
4.3 向浓缩管内管加入50mM Tris (pH 7.5) 至500 μL刻度线处，轻轻吹打混匀。
4.4 重复步骤4.2和4.3四次。
4.5 用移液器从浓缩管中收集叠氮修饰抗体。
4.6 通过测定A280（以A280 = 1.4对应1 mg/mL）来确定抗体浓度(可选)。
注：理论上抗体浓缩管的蛋白回收率>90%，但是部分蛋白样品回收率为60%也属于正常现象。
5 Alkyne、DBCO或DIBO修饰物标记抗体
可使用炔烃修饰试剂盒，将多种探针和标记物连接至叠氮修饰抗体上。具体产品信息见下表。请遵照炔烃标记试剂盒附带的操作规程，完成标记物的连接，并对最终标记抗体进行纯化。
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Glysite is a site‑specific conjugation technology for IgG antibodies, based on Fc glycan remodeling and click chemistry. This technology enables the preparation of stable and homogeneous antibody conjugates from IgG of multiple species and subclasses. Through deglycosylation of the Fc N‑glycans on the antibody, site‑specific conjugation can be achieved via robust click chemistry, resulting in a degree of labeling (DOL) or drug‑to‑antibody ratio (DAR) of 2.
The labeling workflow and principle of this kit are shown in Figure 1. The steps are as follows:
1. Use Antibody concentrator to perform buffer exchange and concentration of the antibody to be labeled.
2. Deglycosylation and introduction of azide groups: The Fc‑specific endoglycosidase EndoS2 hydrolyzes the Fc glycans of various IgG subclasses and species to the innermost GlcNAc moiety. Using β‑1,4‑galactosyltransferase Y289L (GalT), the azide‑containing UDP‑GalNAz is enzymatically attached to the exposed GlcNAc, generating an azide‑activated antibody.
3. Use Antibody concentrator to purify and concentrate the azide‑labeled antibody.
4. Add alkyne‑, DBCO (dibenzocyclooctyne)‑, or DIBO (dibenzylcyclooctyne)‑modified labels such as fluorescent proteins (PE, PE‑Cy5, PE‑Cy7, APC, etc.), fluorophores (Cy3, Cy5, AF488, AF647, etc.), biotin, or amine, and perform a bioorthogonal reaction via strain‑promoted copper‑free click chemistry (SPAAC) to form a stable triazole linkage, completing the antibody labeling.
Each Glysite Duet Antibody Azido Modification Kit contains sufficient reagents for labeling approximately 100–250 μg (G1524532), 1 mg (G1524533), or 5 mg (G1524534) of IgG antibody. The antibody concentrator provided in the kit are used for purification and concentration of the antibody at each step of the labeling workflow (see Figure 1). All operations are carried out under physiological conditions, thus preserving the original quality of the antibody. Since conjugation occurs on the Fc domain, the affinity of the antigen‑binding sites is fully retained.
Figure 1 Schematic diagram of the Glysite Duet Antibody Azido Modification Kit workflow.
Product Features
Contents and storage
Notes: Each component is individually packaged according to its storage temperature (2‑8 °C/-20°C) upon shipment, with differentiated temperature‑controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf‑life is 12 months from the date of manufacture. |
Materials Required (User‑Supplied)
1. Centrifuge.
2. IgG antibody for labeling (free of carrier proteins and azide).
3. Nanodrop or spectrophotometer.
4. 1.5‑2 mL microcentrifuge tubes.
5. Water bath or heating block.
Matters needing attention
1. Bring all kit components to room temperature before use.
2. Before starting the experiment, briefly centrifuge the tubes to ensure all material is at the bottom.
3. Sodium azide must be avoided throughout the entire protocol.
4. Do not reuse Endo S2 Microspin column. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample.
5. This kit does not supply alkyne‑, DBCO‑, or DIBO‑modified fluorophores or other labels.
6. If a chelating agent will be used as a label, it is important to use metal-free water (trace analysis grade) throughout the protocol. The antibody must not be in contact with glass or metal.
7. This product is for professional research use only. It is not intended for clinical diagnosis or treatment, for food or drug use, or for storage in domestic premises.
8. For your safety and health, wear lab coats and disposable gloves when handling reagents.
Instructions for Use
1 Antibody Buffer Exchange and Concentration
1.1 If the antibody is already in 50 mM Tris (pH 7.5) at a concentration of 2–5 mg/mL and the buffer contains no sodium azide, proceed directly to Step 2 (Deglycosylation reaction) without performing this buffer exchange/concentration step.
1.2 Wash the antibody concentrator. Add 0.5 mL of 50 mM Tris (pH 7.5) to a concentrator and centrifuge at 14,000 × g for 5–10 minutes. Discard the liquid from both the inner ultrafiltration tube and the collection tube.
Note: Ensure that the membrane panel of the concentrator faces away from the center of the rotor.
1.3 Transfer 100‑250 μg of the antibody solution to be labeled into the inner tube of the concentrator. Dilute to the 500 μL mark with 50 mM Tris (pH 7.5) and mix gently by pipetting.
1.4 Centrifuge at 14,000 × g for 5–10 minutes. The retentate volume in the inner tube should be ≤100 μL. If it is greater, continue centrifugation in 3‑minute steps until the volume is reduced. Transfer the flow‑through to a new tube and keep it temporarily.
Note: The ultrafiltration inner tube may have a slight leakage. It is recommended to retain the flow‑through until the concentration efficiency is confirmed, then discard it to avoid possible sample loss.
1.5 Add 50 mM Tris (pH 7.5) to the inner tube up to the 500 μL mark and mix gently.
1.6 Repeat steps 1.4 and 1.5 twice more, until the antibody volume is concentrated to 100 μL.
1.7 Transfer the solution from the inner tube to a 1.5 mL centrifuge tube. Determine the antibody concentration by measuring A280 (e.g., A280 = 1.4 corresponds to approximately 1 mg/mL).
Note: The expected protein recovery from the concentrator is >90%, but recovery of ~60% may also be normal for some samples.
2 Deglycosylation: Modification of the N‑Glycan on the Antibody Fc Domain
2.1 Before use, equilibrate the Endo S2 microspin column to room temperature. Break off the bottom cap of the column, place the column in a microcentrifuge tube, and loosen the lid. Centrifuge at 200 × g for 1 min to remove the storage solution. Discard the flow‑through.
2.2 Place the column back in the microcentrifuge tube. Add 300 μL of 50 mM Tris (pH 7.5) onto the resin. Centrifuge at 200 × g for 1 min and discard the flow‑through.
2.3 Repeat step 2.2 two additional times (for a total of three washes).
2.4 Replace the red bottom cap. Immediately add the antibody solution (100 μL) to the column. Seal the column with the lid. Seal the cap and the bottom red cap with Parafilm™ or similar material.
2.5 Fully suspend the resin medium by mixing by inversion, and ensure that the liquid in the column can flow. Incubate the column on a rotating mixer with end‑over‑end mixing at 37 °C for 1 hour.
2.6 Remove the bottom cap, place the column in a new microcentrifuge tube, and loosen the lid. Centrifuge at 1000 × g for 1 min to collect the processed material.
2.7 Replace the red bottom cap, add 100 μL of 50 mM Tris (pH 7.5), and seal the column with the lid. Invert the column several times to mix.
2.8 Remove the bottom cap, place the column in a new microcentrifuge tube, and loosen the lid. Centrifuge at 1000 × g for 1 min to collect the processed material (this is the second elution).
2.9 Combine all collected fractions, including the fraction from step 2.6.
3 Antibody Azido Attachment
3.1 In a 1.5 mL centrifuge tube, add the reagents in the order shown in the table below to prepare the reaction mixture. Vortex to mix, then briefly centrifuge the tube. Seal the cap with Parafilm™ or similar film and incubate overnight at 37°C.
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4 Purification and Concentration of Azido‑Modified Antibody
This step removes excess substrate UDP‑GalNAz. You may also use TBS or other phosphate‑free buffers for purification and collection. The kit provides 50 mM Tris, pH 7.5 for convenience.
4.1 Transfer the enzymatic reaction mixture to the inner tube of the concentrator, add 50 mM Tris (pH 7.5) to the 500 μL mark, and mix gently.
4.2 Centrifuge at 14,000 × g for 5–10 minutes. The retentate volume in the inner tube should be ≤100 μL. If it is greater, repeat centrifugation in 3‑minute steps until the volume is reduced. Discard the flow‑through.
4.3 Add 50 mM Tris (pH 7.5) to the inner tube up to the 500 μL mark and mix gently.
4.4 Repeat steps 4.2 and 4.3 four times.
4.5 Collect the azido‑modified antibody from the concentrator using a pipette.
4.6 (Optional) Determine the antibody concentration by A280 (A280 = 1.4 corresponds to 1 mg/mL).
Note: Expected protein recovery is >90%, but ~60% may be normal for some samples.
5 Labeling of Antibody with Alkyne‑, DBCO‑, or DIBO‑Modified Compounds
Alkyne modification kits are available to attach a variety of probes and labels to the azide modified antibody. For specific product information, see the table below. Follow protocol included with alkyne-modified labeling kit to attach label and purify final labeled antibody.
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| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 1 reaction | 储存 | 单次测试使用量 |
| G1524532WT | Glysite 2位点抗体叠氮基快速标记试剂盒(250μg) (2-8℃组分) | G1524532A | Endo S2 微量离心柱 | 柱体 | 1 EA | 2-8℃， 禁止冷冻 | 每次反应需使用1套 |
| G1524532B | 抗体浓缩管(0.5mL) | 套管 | 2 EA | 2-8℃. | 每次反应需使用2套 | ||
| G1524532DT | Glysite 2位点抗体叠氮基快速标记试剂盒(250μg) (-20℃组分) | G1524532C | 50 mM Tris缓冲液(pH 7.5) | 无色液体 | 20 mL | -20℃. | 请按说明配制 |
| G1524532D | UDP‑GalNAz | 无色液体 | 10 μL | -20℃，避光 | 每次反应需使用10 μL | ||
| G1524532E | 缓冲液添加剂 | 无色液体 | 25 μL | -20℃. | 每次反应需使用25 μL | ||
| G1524532F | GalT(Y289L) | 无色液体 | 10 μL | -20℃. | 每次反应需使用10 μL |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939431 |
分析证书 | G1524532 |
| ZJ26F0939430 |
分析证书 | G1524532 |
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