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亚科因生物研发的V5标签小鼠单克隆抗体（11D5），磁珠偶联（Magnetic Beads Conjugated Anti-V5 Tag Mouse Monoclonal Antibody, 11D5）是一款专为免疫沉淀（IP）设计的高性能工具抗体。该抗体通过共价方式与磁珠偶联，可直接从复杂样品中高效捕获带V5标签的重组蛋白，简化实验流程并缩短操作时间。
V5表位标签源自猴病毒5（SV5）P/V蛋白上的Pk小片段，序列GKPIPNPLLGLDST被广泛用作重组蛋白的检测与纯化标记。本抗体以合成多肽-KLH偶联物免疫小鼠后，经亲和层析纯化获得高特异性IgG1亚型单克隆抗体，再与磁珠共价偶联。磁珠表面高密度抗体（≥1 mg V5抗体/1 ml磁珠）可在磁场作用下快速分离目标蛋白，适用于哺乳动物及细菌表达体系中V5融合蛋白的一步法免疫沉淀。
适用于哺乳动物细胞、细菌裂解液等样本中V5标签融合蛋白的免疫沉淀（IP）、蛋白-蛋白相互作用研究及重组蛋白表达验证。
| 中文名称 | V5标签小鼠单克隆抗体(11D5),磁珠偶联 |
| 英文名称 | Magnetic Beads Conjugated Anti-V5 Tag Mouse Monoclonal Antibody (11D5) |
| 产品货号 | ABT2174 |
| 免疫原 | 合成多肽 |
| 宿主 | 小鼠 |
| 反应性 | 哺乳动物#细菌 |
| 标签 | V5 |
| 检测类型 | IP |
| 克隆类型 | 单克隆 |
| 亚型 | 小鼠IgG1 |
| 纯化工艺 | 通过使用特异性免疫原的亲和层析，从小鼠腹水中纯化抗体。 |
| 偶联物 | 磁珠 |
| 产品形式 | 液体形式 |
| 储存缓冲液 | PBS缓冲液（pH 7.4），含有0.02％叠氮化钠（防腐剂）。 |
| 保存建议 | 从发货之日起，4°C可稳定保存一年。避免反复冻融或者离心。 |
| 运输条件 | 冰袋运输（蓝冰） |
| 警告 | 本文列出的产品仅供研究使用，不适用于人类或临床诊断。我们产品所推荐应用，不是建议使用我们的产品去违反任何专利或许可证。对于使用本产品可能发生的专利侵权或其他违规行为，我们不承担任何责任。 |
Elution Buffer: 0.1 M Glycine-HCl pH 3.0.
Elution Buffer:Neutralization Buffer: 1 M Tris-HCl, pH 8.5.
Neutralization Buffer:Note: Per 500 μL of protein sample add 20 μL Magnetic Beads. Perform the following procedures, according to add 20 μL Magnetic Beads.
Note: Per 500 μL of protein sample add 20 μL Magnetic Beads. Perform the following procedures, according to add 20 μL Magnetic Beads.
Note:(1) Add Magnetic Beads to a 1.5 mL centrifuge tube. Place the centrifuge tube on a Magnetic Separation Rack, let stand for 10 s, remove the supernatant.
(2) Add 1 mL 1×TBS to re-suspend Magnetic Beads, place the tube on a Magnetic Separation Rack, let stand for 10 s, remove the supernatant, repeat 3 times. Add 20 μL 1×TBS to re-suspend Magnetic Beads.
(1) Add Magnetic Beads to a 1.5 mL centrifuge tube. Place the centrifuge tube on a Magnetic Separation Rack, let stand for 10 s, remove the supernatant.
(2) Add 1 mL 1×TBS to re-suspend Magnetic Beads, place the tube on a Magnetic Separation Rack, let stand for 10 s, remove the supernatant, repeat 3 times. Add 20 μL 1×TBS to re-suspend Magnetic Beads.
(1) Add 500 μL protein samples to the processed Magnetic Beads, and incubate at room temperature for 1-2 h or overnight at 4℃ (It is recommended to use vertical rotating mixer with Low-speed rotation).
(2) Place the tube on a Magnetic Separation Rack, let stand for 10 s, remove the supernatant.
(3) Add 1 mL 1×TBS, and re-suspend Magnetic Beads, place the tube on a Magnetic Separation Rack, let stand for 10 s, remove the supernatant, repeat 3-5 times, until OD280 of the supernatant is lesser than 0.05.
(4) Elution
a) Denatured elution: This method is suitable for SDS-PAGE and Western Blotting analysis of elution samples. Add 100 μL (5 times volume of Beads) 1×SDS-PAGE Loading Buffer to the tube and mix well, incubate at 100℃ for 5 min, then Centrifuge at 800 rpm for 1 min, and collect the supernatant to a new tube for SDS-PAGE and Western Blotting analysis.
b) Competitive elution of peptide: This method can maintain their original biological activity, elution can be used for functional analysis. Add 100 μL (5 times volume of Beads) V5 Peptide (0.1-0.2 μg/mL) to the tube and mix well, incubate at 4℃ for 1-2 h (It is recommended to use vertical rotating mixer with Low-speed rotation), then centrifuge at 800 rpm for 2 min at 4℃, and collect the supernatant which is V5-Tag protein and its complex to a new tube. In order to improve the elution efficiency, the incubation time can be Increased or repeat elution. Place V5-Tag protein and its complex on ice to be used, or store at -20℃/-80℃ for long-term. It is recommended to add 100 μL 1×SDS-PAGE Loading Buffer to beads precipitation to test the effect of immunoprecipitation and elution.
c) Acid elution: This method can maintain their original biological activity, elution can be used for functional analysis. Add 100 μL (5 times volume of Beads) Elution Buffer to the tube and mix well, incubate at room temperature for 5-10 min (It is recommended to use vertical rotating mixer with Low-speed rotation), then centrifuge at 800 rpm for 2 min at 4℃, and collect the supernatant which is V5-Tag protein and its complex to a new tube, and immediately add 10 μL Neutralization Buffer to adjust the pH to 7.0-8.0. In order to improve the elution efficiency, elution can be repeated, and combine the same samples. Place His-Tag protein and its complex on ice to be used, or store at -20℃/-80℃ for long-term. It is recommended to add 100 μL 1×SDS-PAGE Loading Buffer to beads precipitation to test the effect of immunoprecipitation and elution.
(1) Add 500 μL protein samples to the processed Magnetic Beads, and incubate at room temperature for 1-2 h or overnight at 4℃ (It is recommended to use vertical rotating mixer with Low-speed rotation).
(2) Place the tube on a Magnetic Separation Rack, let stand for 10 s, remove the supernatant.
(3) Add 1 mL 1×TBS, and re-suspend Magnetic Beads, place the tube on a Magnetic Separation Rack, let stand for 10 s, remove the supernatant, repeat 3-5 times, until OD280 of the supernatant is lesser than 0.05.
(4) Elution
a) Denatured elution: This method is suitable for SDS-PAGE and Western Blotting analysis of elution samples. Add 100 μL (5 times volume of Beads) 1×SDS-PAGE Loading Buffer to the tube and mix well, incubate at 100℃ for 5 min, then Centrifuge at 800 rpm for 1 min, and collect the supernatant to a new tube for SDS-PAGE and Western Blotting analysis.
b) Competitive elution of peptide: This method can maintain their original biological activity, elution can be used for functional analysis. Add 100 μL (5 times volume of Beads) V5 Peptide (0.1-0.2 μg/mL) to the tube and mix well, incubate at 4℃ for 1-2 h (It is recommended to use vertical rotating mixer with Low-speed rotation), then centrifuge at 800 rpm for 2 min at 4℃, and collect the supernatant which is V5-Tag protein and its complex to a new tube. In order to improve the elution efficiency, the incubation time can be Increased or repeat elution. Place V5-Tag protein and its complex on ice to be used, or store at -20℃/-80℃ for long-term. It is recommended to add 100 μL 1×SDS-PAGE Loading Buffer to beads precipitation to test the effect of immunoprecipitation and elution.
c) Acid elution: This method can maintain their original biological activity, elution can be used for functional analysis. Add 100 μL (5 times volume of Beads) Elution Buffer to the tube and mix well, incubate at room temperature for 5-10 min (It is recommended to use vertical rotating mixer with Low-speed rotation), then centrifuge at 800 rpm for 2 min at 4℃, and collect the supernatant which is V5-Tag protein and its complex to a new tube, and immediately add 10 μL Neutralization Buffer to adjust the pH to 7.0-8.0. In order to improve the elution efficiency, elution can be repeated, and combine the same samples. Place His-Tag protein and its complex on ice to be used, or store at -20℃/-80℃ for long-term. It is recommended to add 100 μL 1×SDS-PAGE Loading Buffer to beads precipitation to test the effect of immunoprecipitation and elution.
a) Denatured elution: This method is suitable for SDS-PAGE and Western Blotting analysis of elution samples. Add 100 μL (5 times volume of Beads) 1×SDS-PAGE Loading Buffer to the tube and mix well, incubate at 100℃ for 5 min, then Centrifuge at 800 rpm for 1 min, and collect the supernatant to a new tube for SDS-PAGE and Western Blotting analysis.
b) Competitive elution of peptide: This method can maintain their original biological activity, elution can be used for functional analysis. Add 100 μL (5 times volume of Beads) V5 Peptide (0.1-0.2 μg/mL) to the tube and mix well, incubate at 4℃ for 1-2 h (It is recommended to use vertical rotating mixer with Low-speed rotation), then centrifuge at 800 rpm for 2 min at 4℃, and collect the supernatant which is V5-Tag protein and its complex to a new tube. In order to improve the elution efficiency, the incubation time can be Increased or repeat elution. Place V5-Tag protein and its complex on ice to be used, or store at -20℃/-80℃ for long-term. It is recommended to add 100 μL 1×SDS-PAGE Loading Buffer to beads precipitation to test the effect of immunoprecipitation and elution.
c) Acid elution: This method can maintain their original biological activity, elution can be used for functional analysis. Add 100 μL (5 times volume of Beads) Elution Buffer to the tube and mix well, incubate at room temperature for 5-10 min (It is recommended to use vertical rotating mixer with Low-speed rotation), then centrifuge at 800 rpm for 2 min at 4℃, and collect the supernatant which is V5-Tag protein and its complex to a new tube, and immediately add 10 μL Neutralization Buffer to adjust the pH to 7.0-8.0. In order to improve the elution efficiency, elution can be repeated, and combine the same samples. Place His-Tag protein and its complex on ice to be used, or store at -20℃/-80℃ for long-term. It is recommended to add 100 μL 1×SDS-PAGE Loading Buffer to beads precipitation to test the effect of immunoprecipitation and elution.
Note: a) For a few samples, due to differences in target proteins, the binding of V5-Tag and Anti-V5 antibody is very strong, and the effect of Acid elution and Competitive elution of peptide may be poor. Therefore, SDS-PAGE Loading Buffer denaturation elution method is recommended as a priority; b) Due to the difference of target protein, the elution efficiency of acid elution method also varies to some extent. If the requirement of elution efficiency is high, the pH value of acidic eluent can be adjusted appropriately between 2.5-3.1, and the pH value or quantity of corresponding neutralizing solution should be adjusted appropriately. For example, 100 μL Acid Elution Buffer (0.1 M Glycine-HCl, pH 2.8) and 15 μL Neutralizing Buffer (1 M Tris-HCl, pH 8.5).
Note: a) For a few samples, due to differences in target proteins, the binding of V5-Tag and Anti-V5 antibody is very strong, and the effect of Acid elution and Competitive elution of peptide may be poor. Therefore, SDS-PAGE Loading Buffer denaturation elution method is recommended as a priority; b) Due to the difference of target protein, the elution efficiency of acid elution method also varies to some extent. If the requirement of elution efficiency is high, the pH value of acidic eluent can be adjusted appropriately between 2.5-3.1, and the pH value or quantity of corresponding neutralizing solution should be adjusted appropriately. For example, 100 μL Acid Elution Buffer (0.1 M Glycine-HCl, pH 2.8) and 15 μL Neutralizing Buffer (1 M Tris-HCl, pH 8.5).
Note:| Catalog No. | Product Name |
|---|---|
| ABT2014 | Magnetic Beads Conjugated Anti-DDDDK Tag Mouse Monoclonal Antibody (1B10) |
| ABT2024 | Magnetic Beads Conjugated Anti-GFP Tag Mouse Monoclonal Antibody (3D3) |
| ABT2044 | Magnetic Beads Conjugated Anti-HA Tag Mouse Monoclonal Antibody (4F6) |
| ABT2054 | Magnetic Beads Conjugated Anti-His Tag Mouse Monoclonal Antibody (5C3) |
| ABT2064 | Magnetic Beads Conjugated Anti-Myc Tag Mouse Monoclonal Antibody (2D5) |
The reagent is only used in the field of scientific research, not suitable for clinical diagnosis or other purposes.
A: 荧光western blotting的结果需要特殊的仪器检测，即一种荧光成像仪器。即运用荧光成像仪可以对荧光WB的实验结果进行成像和数据采集分析。如果希望应用荧光成像做WB，建议选用Abbkine DyLight 680和DyLight 800荧光二抗，货号分别为A23710、A23720、A23910、A23920。
A: 0.5 mg/ml。
A: 甘油是可以减慢抗体分子之间的碰撞，起到抗体保护剂的作用。
请等待最新文献信息更新。
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