Rb69 gene 32 protein 是 Rb69 噬菌体基因 32 编码的单链 DNA结合蛋白，分子量为 34 KDa， 为 Rb69 噬菌体 DNA 复制和修复所必需物质。它协调性地结合并稳定瞬时形成的单链 DNA 区域，在 Rb69噬菌体 DNA 复制过程中起着重要的结构性作用。该蛋白也被广泛地用于稳定和标记单链区域，以便用电子显微镜观察细胞内 DNA 的结构。 Rb69 gene 32 protein 可以促进限制性内切酶的消化反应、 RT- PCR 中反转录的效率、增强 DNA 聚合酶的活性， 也可以被用于重组酶聚合酶扩增 (RPA) 反应。
应用方向
1. 在 RT-PCR 过程中，增加反转录的产量和延伸能力；
2. 土壤样品进行 PCR 时，增加目的片段的产量和特异性；
3. 稳定和标记 ssDNA 结构。
质量控制
1. 蛋白纯度检测
使用 SDS-PAGE 凝胶电泳检测，蛋白纯度不低于 95%。
2. 核酸内切酶活性检测
将 10 μg Rb69 gene 32 protein 与 200 ng 超螺旋质粒 DNA 在37℃下，共同温育 4 h 后，使用琼脂糖凝胶电泳检测，少于 10% 的质粒 DNA 转变成缺刻或线性状态。
3. 非特异性核酸酶活性检测
将 10 μg Rb69 gene 32 protein 与 15 ng 双链 DNA 片段在 37 ℃温育 16 h，使用琼脂糖凝胶电泳检测双链 DNA 底物无变化。
4. RNase 活性检测
将 10 μg Rb69 gene 32 protein 与 500 ng 总 RNA 在 37 ℃温育1 h，使用琼脂糖凝胶电泳检测超过 90% 的 RNA 仍保持完整。
5. 宿主 DNA 残留检测
使用大肠杆菌 16S rDNA 特异性引物探针组， 采用荧光定量 PCR法检测 10 μg Rb69 gene 32 protein，大肠杆菌宿主基因组 DNA 残留低于 10 copies。
失活条件
65 ℃孵育 20 min 可使该蛋白失活。
Rb69 gene 32 protein is a single-stranded DNA-binding protein encoded by gene 32 of Rb69 phage, with a molecular weight of 34 kDa. It is essential for DNA replication and repair of Rb69 phage. The protein binds cooperatively and stabilizes transiently formed single-stranded DNA regions, playing an important structural role during Rb69 phage DNA replication. It is also widely used to stabilize and label single-stranded DNA regions for observing intracellular DNA structure under electron microscopy. Rb69 gene 32 protein can enhance restriction endonuclease digestion efficiency, improve reverse transcription efficiency in RT-PCR, stimulate DNA polymerase activity, and is applicable to Recombinase Polymerase Amplification (RPA) reactions.
Applications
- Increase reverse transcription yield and extension capacity in RT-PCR.
- Improve target fragment yield and specificity in PCR of soil samples.
- Stabilize and label ssDNA structures.
Quality Control
1. Protein Purity
Determined by SDS-PAGE; purity ≥ 95%.
2. Endonuclease Activity Assay
10 μg of Rb69 gene 32 protein was incubated with 200 ng supercoiled plasmid DNA at 37 °C for 4 h. Agarose gel electrophoresis showed less than 10% of plasmid DNA was converted into nicked or linear form.
3. Non-specific Nuclease Activity Assay
10 μg of the protein was incubated with 15 ng double-stranded DNA fragment at 37 °C for 16 h. No alteration of dsDNA substrate was observed by agarose gel electrophoresis.
4. RNase Activity Assay
10 μg of the protein was incubated with 500 ng total RNA at 37 °C for 1 h. Agarose gel electrophoresis confirmed over 90% of RNA remained intact.
5. Host Genomic DNA Residue
Quantitative real-time PCR with E. coli 16S rDNA-specific primers and probes was used to detect 10 μg protein; residual E. coli genomic DNA was less than 10 copies.
Inactivation Condition
Incubation at 65 °C for 20 min completely inactivates the protein.
ZJ26F0535930
危险品化学品经营许可证（带存储）