Gelatin-Zymography Kit – 明胶酶谱分析用试剂盒说明书
Introduction
Gelatin-Zymography Kit
Matrix metalloproteinases (MMPs) belong to the family of metalloproteinases, which consists of at least 20 members, and known to be involved in the metabolism of extracellular matrix proteins. MMPs are widely detected by zymography. The Gelatin-Zymography Kit (Cat.No.AK47) provides an easy system of the electrophoresis for zymography. This product is used for detecting ProMMP-2, MMP-2 and ProMMP-9 in blood, body fluid, secretion, cell lysate, cell culture medium, and other samples.
基质金属蛋白酶 (MMP) 属于金属蛋白酶家族，该家族由至少 20 个成员组成，并且已知参与细胞外基质蛋白的代谢。 MMP 可通过酶谱法广泛检测。 Gelatin-Zymography Kit (Cat.No.AK47) 提供了一个简单的酶谱电泳系统。 本产品用于检测血液、体液、分泌物、细胞裂解液、细胞培养基等样品中的ProMMP-2、MMP-2、ProMMP-9。
Components/Storage
| Component |
Quantity |
Storage |
| Precast gel for 12 samples |
5 pieces |
4ºC |
| Electrophoresis Buffer (×10) |
100 mL × 2 |
4ºC |
| Washing Buffer (×10) |
100 mL × 1 |
4ºC |
| Reaction Buffer (×10) |
25 mL × 1 |
4ºC |
| Sample Preparation Buffer |
5 mL × 1 |
4ºC |
| Staining Solution |
100 mL × 1 |
room temperature |
| MMP markers (ProMMP-2, MMP-2, ProMMP-9) |
0.2 mL |
4ºC |
The size of the glass plate gel is 100(W)×100(H) and the thickness is 1mm. One kit contains regents for 60 samples
玻璃板凝胶的尺寸为 100(W)×100(H)，厚度为 1mm。 一个试剂盒包含 60 个样品的试剂
Materials required but not provided
・acetic acid ・methanol ・distilled water
需要但未提供的材料
・乙酸・甲醇・蒸馏水
Preparation of reagents
・Electrophoresis Buffer
Dilute Electrophoresis Buffer (×10) with distilled water to a 1x concentrated solution.
Note: Diluted buffer should be stored at 4°C.
试剂的制备
・电泳缓冲液
用蒸馏水稀释电泳缓冲液 (×10) 至 1x 浓缩溶液。
注意：稀释的缓冲液应储存在 4°C。
・Washing Buffer
Dilute Washing Buffer (×10) with distilled water to a 1x concentrated solution.
Note: Diluted buffer should be stored at 4°C.
・洗涤缓冲液
用蒸馏水稀释洗涤缓冲液 (×10) 至 1x 浓缩溶液。
注意：稀释的缓冲液应储存在 4°C。
・Reaction Buffer
Dilute Reaction Buffer (×10) with distilled water to a 1x concentrated solution.
Note: Diluted buffer should be stored at 4°C.
・反应缓冲液
用蒸馏水稀释反应缓冲液 (×10) 至 1x 浓缩溶液。
注意：稀释的缓冲液应储存在 4°C。
・De-staining Solution
This solution is not included in this kit. Mix Acetic acid: Methanol: Distilled water = 5 : 30 : 65.
Note: De-staining Solution should be stored at Room Temperature.
・脱色液
此解决方案不包含在此套件中。 混合乙酸：甲醇：蒸馏水 = 5：30：65。
注意：脱色溶液应在室温下储存。
Protocol
- Load100-150 mL of Electrophoresis Buffer to the lower chamber (anode). Refer to the instruction manual of your electrophoresis tank/chamber to know appropriate volume of the buffer.
- Takeout the comb on precast gel carefully and set the gel to electrophoresis The side of sample holes should be set on the upper side. If the sample holes are disturbed, fix them up with needle etc.
- Loadaround 100 mL of Electrophoresis Buffer to the upper chamber (cathode).
- Mix samples with equivalent volume of sample preparation buffer. Incubate for 15 minutes at Room Temperature. (Do NOT heat the samples.)
- Apply the samples and MMP markers to gel plate. (Fig.1: 10ul of MMP markers applied and enzymatic reacted for 24 hrs. at 37°C)Run electrophoresis at 15 mA constant current. (If you use 2 gels, set the current at 30 mA)
1、将 100-150 mL 的电泳缓冲液装入下室（阳极）。 请参阅您的电泳槽/室的说明手册以了解适当的缓冲液体积。
2、小心取出预制凝胶上的梳子，将凝胶置于电泳位置。样品孔的一侧应置于上侧。 如果样品孔受到干扰，请用针等固定它们。
3、将 100 mL 的电泳缓冲液加载到上腔室（阴极）。
4、将样品与等量的样品制备缓冲液混合。 在室温下孵育 15 分钟。 （不要加热样品。）
5、将样品和 MMP 标记应用于凝胶板上。 （图 1：应用 10ul MMP 标记物并在 37°C 下酶促反应 24 小时）在 15 mA 恒定电流下运行电泳。 （如果您使用 2 块凝胶，请将电流设置为 30 mA）
*MMP markers can be used without sample preparation buffer.
- Afterthe run is completed, turn off the electrophoresis And take out the gel plate from the chamber.
- Removethe upper glass plate of the gel plate and peel off the gel carefully with a
- Putthe gel in the tray with 200 mL of Washing Incubate with shaking at Room Temperature.
- Putthe gel in the container with 50 mL of Reaction Buffer and seal up the Incubate the gel in the container in incubator at 37°C for 20-40 hrs (Lower enzyme concentration needs longer reaction time.).
- After enzymatic reaction, put the gel in the container with Staining solution. Incubate for 30 minutes at Room Temperature to stain protein.
- Putthe gel in the container with De-staining And incubate for 30minutes – several hours to de-stain.
运行完成后，关闭电泳并从腔室中取出凝胶板。
取下凝胶板的上玻璃板并用
将凝胶放入装有 200 mL 洗涤液的托盘中，在室温下摇动孵育。
将凝胶放入装有 50 mL 反应缓冲液的容器中并密封。将容器中的凝胶在 37°C 的培养箱中孵育 20-40 小时（较低的酶浓度需要较长的反应时间。）。
酶促反应后，将凝胶放入装有染色液的容器中。 在室温下孵育 30 分钟以染色蛋白质。
将凝胶放入带有脱色剂的容器中并孵育 30 分钟 – 数小时以脱色。
Application example
Fig. 1 10ul of MMP markers applied and enzymatic reacted for 24 hrs. at 37°C
References 参考文献
- Miyazaki, K., Ohta, Y., Nagai, M., Morimoto, N., Kurata, T., Takehisa, Y., Ikeda, Y., Matsuura, T., Abe, K. Disruption of Neurovascular Unit Prior to Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis. J. Neurosci. Res. 89, 718-728 (2011)
- Fujiwara,, Kashima, T. G., Kunita, A., Kii, I., Komura, D., Grigoriadis, A. E., Kudo, A., Aburatani, H., Fukayama, M. Stable Knockdown of S100A4 Suppresses Cell Migration and Metastasis of Osteosarcoma. Tumour Biol.
- Aoki, T., Kataoka, H., Ishibashi, R., Nozaki, K., Hashimoto, N. Simvastatin Suppresses the Progression of Experimentally Induced Cerebral Aneurysms in Rats. Stroke. 39, 1276-85 (2008)
- Kuramochi, D., Unoki, H., Bujo, H., Kubota, Y., Jiang, M., Rikihisa, N., Udagawa, A., Yoshimoto, S., Ichinose, M.,Saito, Y. Matrix Metalloproteinase 2 Improves The TransplantedAdipocyte Survival in Mice. Eur. J. Clin. Invest. 38, 752-759 (2008)
- Fujimoto,, Takagi, Y., Aoki, T., Hayase, M., Marumo, T., Gomi, M., Nishimura, M., Kataoka, H., Hashimoto, N., Nozaki, K. Tissue Inhibitor of Metalloproteinases Protect Blood-brain Barrier Disruption in Focal Cerebral Ischemia. J. Cereb. Blood Flow Metab. 28, 1674-1685 (2008)
Notice to Purchaser
This product has a short expiration date (less than 6 months). Please use it after arrival as soon as possible.
This product is to be used for Research Purposes Only. It is not to be used for Drug or Diagnostic Purposes, nor is it intended for Human Use. Primary cell products may not be resold, modified for resale, or used to manufacture commercial products without the express written consent of Primary Cell Co., Ltd.
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买家须知
此产品的有效期很短（少于 6 个月）。 请在抵达后尽快使用。
本产品仅用于研究目的。 它不用于药物或诊断目的，也不用于人类使用。 未经 Primary Cell Co., Ltd. 的明确书面同意，不得转售、改装转售或用于制造商业产品。
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