BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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中生成稳定、均一的抗体偶联物，所得标记度（DOL）或药物抗体比（DAR）为4。通过酶促反应定点对IgG抗体Fc区域高度保守N297位糖基化修饰进行叠氮基(Azide)标记，经叠氮修饰的抗体随后可通过无铜点击反应，与炔基(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)等共价连接，且无需对蛋白质进行还原处理。这使您能够根据实验需要，为抗体选择不同的荧光标记，通过链霉亲和素连接其他分子，或利用叠氮反应性基团偶联您自有的分子。
每套Glysite 4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 (G1524535)、1mg (G1524536) 和5mg (G1524537) 的IgG抗体或N-连接型糖基化修饰的蛋白。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 4位点抗体叠氮基快速标记试剂盒原理图
产品特点
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所需自备材料
1. 离心机
2. 用于标记的IgG抗体
3. Nanodrop或分光光度计
4. 2mL离心管
5. 恒温水浴锅或金属浴
注意事项
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. 本试剂盒不提供Alkyne、DBCO或DIBO修饰的荧光基团等标记物。
5. 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。
6. 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10mg/mL，且缓冲液中不含叠氮化钠，可直接进行步骤2的酶促反应，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12mL 50mM Tris (pH 7.5) ，3500 × g离心10-15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5mg 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12mL刻度线处，轻轻吹打混匀。
1.4 3500 × g离心10-15 分钟，内管滞留液体积应不超过0.5mL。若滞留液体积大于 0.5mL ，则重复离心，每次5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12mL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0.5mL。
1.7 将超滤内管中的溶液转移至2mL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 抗体糖链修饰与叠氮基团连接
2.1 按下表在2mL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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3 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。
3.1 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12mL刻度线处，轻轻吹打混匀。
3.2 3500 × g离心10-15 分钟，内管滞留液体积应不超过0.5mL。若滞留液体积大于0.5mL，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。
3.3 向浓缩管内管加入50mM Tris (pH 7.5) 至12mL刻度线处，轻轻吹打混匀。
3.4 重复步骤3.2和3.3四次。
3.5 用移液器从浓缩管中收集叠氮修饰抗体。
3.6 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。
4 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。
| Catalog No. | Product |
| G1528096 | GIySite 生物素抗体标记试剂盒(5mg) |
| G1528097 | GIySite AF488 抗体标记试剂盒(5mg) |
| G1528098 | GIySite AF594 抗体标记试剂盒(5mg) |
| G1528099 | GIySite AF647 抗体标记试剂盒(5mg) |
| G1528100 | GIySite R-PE 抗体标记试剂盒(5mg) |
| G1528101 | GIySite APC 抗体标记试剂盒(5mg) |
| G1528102 | GIySite PE-Cy5 抗体标记试剂盒(5mg) |
| G1528103 | GIySite PE-Cy7 抗体标记试剂盒(5mg) |
| G1528104 | GIySite APC-AF750 抗体标记试剂盒(5mg) |
| G1528105 | GIySite PerCP 抗体标记试剂盒(5mg) |
| G1528106 | GIySite PerCP-Cy5.5 抗体标记试剂盒(5mg) |
Glysite is a site‑specific conjugation technology for IgG antibodies, based on Fc glycan remodeling and click chemistry. The technology generates stable and homogeneous antibody conjugates from several species and subclasses, with a degree of labeling (DOL) or drug‑to‑antibody ratio (DAR) of 4. Through an enzymatic reaction, an azido moiety is site‑specifically introduced onto the highly conserved N‑glycan at position N297 of the IgG Fc region. The azido‑modified antibody can then be covalently linked, via a copper‑free click reaction, to alkyne‑, DBCO (dibenzocyclooctyne)‑, or DIBO (dibenzylcyclooctyne)‑modified fluorescent proteins (PE, PE‑Cy5, PE‑Cy7, APC, etc.), fluorophores (Cy3, Cy5, AF488, AF647, etc.), biotin, amine, or other molecules, without the need for protein reduction. This provides the flexibility to choose different fluorescent labels for your antibody, to attach other molecules via streptavidin, or to conjugate your own azido‑reactive moieties according to your assay needs.
Each Glysite Quartet Antibody Azido Modification Kit contains sufficient reagents to label approximately 100‑250 μg (G1524535), 1 mg (G1524536), or 5 mg (G1524537) of IgG antibody or N‑linked glycoproteins. The antibody concentrators provided in the kit are used to purify and concentrate the antibody at each step of the labeling workflow (see Figure 1). All steps are performed under physiological conditions, thus preserving the quality of the antibody. Because the conjugation occurs on the Fc domain, the affinity of the antigen‑binding sites is fully retained.
Figure 1 Schematic diagram of the Glysite Quartet Antibody Azido Modification Kit workflow.
Product Features
Contents and storage
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Materials Required (User‑Supplied)
1. Centrifuge
2. IgG antibody for labeling
3. NanoDrop or spectrophotometer
4. 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. This kit does not supply alkyne‑, DBCO‑, or DIBO‑modified fluorophores or other labels.
5. 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.
6. 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 10 mg/mL and the buffer contains no sodium azide, proceed directly to Step 2 (enzymatic reaction) without performing this buffer exchange/concentration step.
1.2 Wash the antibody concentrator. Add 12 mL of 50 mM Tris (pH 7.5) to a concentrator and centrifuge at 3500 × g for 10-15 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 5 mg of the antibody solution to be labeled into the inner tube of the concentrator. Dilute to the 12 mL mark with 50 mM Tris (pH 7.5) and mix gently by pipetting.
1.4 Centrifuge at 3500 × g for 10-15 minutes. The retentate volume in the inner tube should be ≤0.5 mL. If it is greater, continue centrifugation in 5‑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 12 mL mark and mix gently.
1.6 Repeat steps 1.4 and 1.5 twice more, until the antibody volume is concentrated to 0.5 mL.
1.7 Transfer the solution from the inner tube to a 2 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 Antibody Glycan Modification and Azido Attachment
2.1 In a 2 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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3 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.
3.1 Transfer the enzymatic reaction mixture to the inner tube of the concentrator, add 50 mM Tris (pH 7.5) to the 12 mL mark, and mix gently.
3.2 Centrifuge at 3500 × g for 10-15 minutes. The retentate volume in the inner tube should be ≤0.5 mL. If it is greater, repeat centrifugation in 5‑minute steps until the volume is reduced. Discard the flow‑through.
3.3 Add 50 mM Tris (pH 7.5) to the inner tube up to the 12 mL mark and mix gently.
3.4 Repeat steps 3.2 and 3.3 four times.
3.5 Collect the azido‑modified antibody from the concentrator using a pipette.
3.6 (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.
4 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.
| Catalog No. | Product |
| G1528096 | GIySite Biotin Antibody Labeling Kit (5mg) |
| G1528097 | GIySite AF488 Antibody Labeling Kit (5mg) |
| G1528098 | GIySite AF594 Antibody Labeling Kit (5mg) |
| G1528099 | GIySite AF647 Antibody Labeling Kit (5mg) |
| G1528100 | GIySite R-PE Antibody Labeling Kit (5mg) |
| G1528101 | GIySite APC Antibody Labeling Kit (5mg) |
| G1528102 | GIySite PE-Cy5 Antibody Labeling Kit (5mg) |
| G1528103 | GIySite PE-Cy7 Antibody Labeling Kit (5mg) |
| G1528104 | GIySite APC-AF750 Antibody Labeling Kit (5mg) |
| G1528105 | GIySite PerCP Antibody Labeling Kit (5mg) |
| G1528106 | GIySite PerCP-Cy5.5 Antibody Labeling Kit (5mg) |
| Storage Unit No. | 名称 | 组分 | 组分名称 | 物理外观 | 1 reaction | 储存 | 单次测试使用量 |
| G1524537DT | Glysite 4位点抗体叠氮基快速标记试剂盒(5mg) (-20℃组分) | G1524537A | 50 mM Tris缓冲液(pH 7.5) | 无色液体 | 100 mL | -20℃. | 请按说明配制 |
| G1524537B | UDP‑GalNAz | 无色液体 | 100 μL | -20℃，避光 | 每次反应需使用100 μL | ||
| G1524537C | 缓冲液添加剂 | 无色液体 | 100 μL | -20℃. | 每次反应需使用100 μL | ||
| G1524537D | 酶混合物 | 无色液体 | 100 μL | -20℃. | 每次反应需使用100 μL | ||
| G1524537NT | Glysite 4位点抗体叠氮基快速标记试剂盒(5mg) (常温组分) | G1524537E | 抗体浓缩管(15mL) | 套管 | 2 EA | 室温 | 每次反应需使用2套 |
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 |
|---|---|---|
 ZJ26F0939184 |
分析证书 | G1524537 |
| ZJ26F0939185 |
分析证书 | G1524537 |
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