Gelatin zymography kit (ATTO type)
||Zymography Kit 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.
||Precast gel for 12 samples, 5 pieces
Electrophoresis Buffer (x10), 100 ml x 2
Washing Buffer (x10), 100 ml
Reaction Buffer (x10), 25 ml
Sample Preparation Buffer, 5 ml
Staining Solution, 100 ml
MMP markers (ProMMP-2, MMP-2, ProMMP-9), 0.2 ml
The size of the glass plate gel is 120 (W) x 100 (H) and the thickness is 1 mm , for ATO electrophoresis system.
This product has a short expiration date (less than 3 months). Please use it after arrival as soon as possible.
1. Load 100-150 ml of Electrophoresis Buffer to the lower chamber (anode). Refer to the instruction manual ofyour electrophoresis tank/chamber to know appropriate volume of the buffer.
2. Take out the comb on precast gel carefully and set the gel to electrophoresis chamber. The side of sample holes should be set on the upper side. If the sample holes aredisturbed, fix them up with needle etc.
3. Load around 100 ml of Electrophoresis Buffer to the upper chamber (cathode).
4. Mixsamples with equivalent volume of sample preparation buffer. Incubate for 15 minutes at Room Temperature. (Do NOT heat thesamples.)
*MMP markers can be used without sample preparation buffer.
5. Apply the samples and MMP markers to gel plate.
6.Run electrophoresis at 15 mA constant current. (If you use 2 gels, set the current at 30 mA)
7. After the run is completed, turn off the electrophoresis chamber, and take out the gel plate from the chamber.
8. Remove the upper glass plate of thegel plate and peel off the gel carefully with a spatula.
9. Put the gel in the tray with 200 ml of Washing Buffer. Incubate withshaking at Room Temperature.
10. Put the gel in the container with 50 ml of Reaction Buffer and seal up the container. Incubate the gel in the container in incubator at 37C for 20-40 hrs (Lower enzyme concentration needs longer reaction time.)
11. After enzymatic reaction, put the gel in the container with Staining solution. Incubate for 30 minutes at Room Temperature to stain protein.
12. Put the gel in the container with De-staining solution. And incubate for 30 minutes to several hourstode-stain.
• Miyazaki, K., Ohta, Y., Nagai, M., Morimoto, N., Kurata, T., Takehisa, Y., Ikeda, Y., Matsuura, T., Abe, K. J. Neurosci. Res. 89, 718-728 (2011)
• Fujiwara, M., Kashima, T. G., Kunita, A., Kii, I., Komura, D., Grigoriadis, A. E., Kudo, A., Aburatani, H., Fukayama, M. Tumour Biol.
• Aoki, T.,Kataoka, H., Ishibashi, R., Nozaki, K., Hashimoto, N. 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. Eur. J. Clin. Invest. 38, 752-759 (2008)
• Fujimoto, M., Takagi, Y., Aoki, T., Hayase, M., Marumo, T., Gomi, M., Nishimura, M., Kataoka, H., Hashimoto, N., Nozaki, K. J. Cereb. Blood Flow Metab. 28, 1674-1685 (2008)