O-内切糖苷酶，大肠杆菌中重组表达，来源于 Streptococcus pneumoniae，该酶具有高度特异性，可从单独的丝氨酸或苏氨酸残基或作为糖肽或糖蛋白的一部分释放 Galβ1-3GalNAc。对丝氨酸和苏氨酸没有明显的偏好。应用于糖蛋白的生物合成分析,O -聚糖的生物活性蛋白质 O -糖基化检测和结合位点分析。
O-Glycosidase 作用 O-连二糖核心的类型
组分和说明
| O755132 | Component | 1KU | 5KU | Storage | | O755132A | O-Glycosidase | 1KU | 5KU | -20℃. Avoid freeze/thaw cycle. | | O755132B | 10×Glycoprotein Denaturing Buffer (0.5% SDS, 40mM DTT) | 200μl | 1000μl | -20℃. Avoid freeze/thaw cycle. | | O755132C | 10×GlycoBuffer 2 (500 mM Sodium Phosphate, pH7.5) | 200μl | 1000μl | -20℃. Avoid freeze/thaw cycle. | | O755132D | 10% NP-40 | 200μl | 1000μl | -20℃. Avoid freeze/thaw cycle. |
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使用前准备：
使用前，请将O-Glycosidase 试剂取出，冰上或者4°C解冻，10000rpm 离心10 秒，确保所有试剂都在管底。
反应试剂：将-20℃储存的 10×Glycoprotein Denaturing Buffer 缓冲液、10×GlycoBuffer 2 及 10%NP-40 溶液取出，待用。
注意：根据实验需要，准备相应的试剂，如非变性条件下进行去糖基化反应，不需要准备10×Denaturing 缓冲液及10% NP-40 溶液。
使用方法：
1）变性条件下糖蛋白去糖基化
1、用去离子水溶解 1-20 µg 的糖蛋白，加入 1 µl 的 10×Glycoprotein Denaturing Buffer，用去离子水定容到 10 µl；
2、75 ℃孵育 10 min；
3、加入 2 µl 的 10×GlycoBuffer 2，2 µl 的 10% NP-40，轻轻吹打混匀；
4、加入 1-4 µl 的 O-Glycosidase，加去离子水到 20 µl，轻轻吹打混匀；
5、37℃孵育 1-4hr；
6、用于 SDS-PAGE 分析或 HPLC 分析。
2) 非变性条件下糖蛋白去糖基化
1. 取 10-100µg 糖蛋白溶液，加入 2µl 10×GlycoBuffer 2，2-5µl O-Glycosidase ，补加纯化水使得反应体系总体积为 20µl，轻柔混匀；
2. 37℃条件下反应 4-24hr。
注意：当对天然糖蛋白去糖基化时，建议将等量糖蛋白样品进行变性后再同步进行酶切实验作为阳性对照，以确定非变性条件下去糖基化反应的程度。
【注】体系放大时候，孵育时间或酶量需要根据实际情况调整，可低速震荡混匀，可以增加转化率。
注意事项
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Endo-O-glycosidase, recombinantly expressed in Escherichia coli, derived from Streptococcus pneumoniae. This enzyme exhibits high substrate specificity and releases Galβ1-3GalNAc from isolated serine or threonine residues, as well as from serine/threonine residues within glycopeptides or glycoproteins. It shows no significant preference for serine over threonine or vice versa. It is applied in biosynthesis analysis of glycoproteins, detection of O-glycosylation on bioactive proteins, and mapping of O-glycan binding sites.
Types of O-linked disaccharide cores targeted by O-Glycosidase
Components List
| O755132 | Component | 1KU | 5KU | Storage | | O755132A | O-Glycosidase | 1KU | 5KU | -20℃. Avoid freeze/thaw cycle. | | O755132B | 10×Glycoprotein Denaturing Buffer (0.5% SDS, 40mM DTT) | 200μl | 1000μl | -20℃. Avoid freeze/thaw cycle. | | O755132C | 10×GlycoBuffer 2 (500 mM Sodium Phosphate, pH7.5) | 200μl | 1000μl | -20℃. Avoid freeze/thaw cycle. | | O755132D | 10% NP-40 | 200μl | 1000μl | -20℃. Avoid freeze/thaw cycle. |
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Preparations before use
Before use, take out the O-Glycosidase reagent, thaw it on ice or at 4°C, and centrifuge at 10,000 rpm for 10 seconds to ensure all reagents are collected at the bottom of the tube.
Reaction reagents
Retrieve the following reagents stored at −20 °C for standby use: 10× Glycoprotein Denaturing Buffer, 10× GlycoBuffer 2 and 10% NP-40 solution.Note: Prepare reagents as required by your experiment. The 10× Denaturing Buffer and 10% NP-40 solution are not required if deglycosylation is performed under native (non-denaturing) conditions.
Operating Procedures
1) Deglycosylation of glycoproteins under denaturing conditions
1. Dissolve 1–20 μg glycoprotein in deionized water, add 1 μL 10× Glycoprotein Denaturing Buffer, and bring the total volume to 10 μL with deionized water.
2. Incubate at 75 °C for 10 min.
3. Add 2 μL 10× GlycoBuffer 2 and 2 μL 10% NP-40, mix gently by pipetting up and down.
4. Add 1–4 μL O-Glycosidase, top up with deionized water to a final volume of 20 μL, and mix gently by pipetting.
5. Incubate at 37 °C for 1–4 hours.
6. Subject the sample to SDS-PAGE or HPLC analysis.
2) Deglycosylation of glycoproteins under non-denaturing conditions
1. Take 10–100 μg glycoprotein solution, add 2 μL 10× GlycoBuffer 2 and 2–5 μL O-Glycosidase, then add purified water to make a total reaction volume of 20 μL. Mix gently.
2. Carry out the reaction at 37 °C for 4–24 hours.
Note: When deglycosylating native glycoproteins, it is recommended to prepare an equal amount of the same glycoprotein sample, denature it and perform parallel enzymatic digestion as a positive control to assess the extent of deglycosylation under non-denaturing conditions.
Remark: When scaling up the reaction system, adjust incubation time or enzyme dosage as appropriate. Gentle low-speed shaking can improve deglycosylation efficiency.
Precautions
Wear a lab coat and disposable gloves during operation for personal safety and health. For research use only.
ZJ25F0724134
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