Ordering Information TOP

Frequently Asked Questions (FAQ) TOP

Zymography TOP

• [01] What is zymography?
• [02] Why can proMMP without enzyme activity be detected as a band by zymography?
• [03] How long does it take to run with a constant current of 15mA?
• [04] How do I take a good picture of the gels?
• [05] How much protein should I apply per lane?
• [06] How do I extract MMPs from tissues?
• [07] What should I avoid during sample processing?
• [08] What are the effects of anticoagulants used in blood collection?
• [09] What are the sizes of the precast gels included in the Gelatin Casein Zymo Electrophoresis Kit?

Gelatin Zymography TOP

• [01] What is the concentration of the precast gels?
• [02] What is the band detected between proMMP2 and MMP2?
• [03] Is the mobility of proMMP9 in mice and humans different?
• [04] What are the bands detected on the polymer side of proMMP9?

Casein Zymography TOP

• [01] What is the concentration of the precast gels?

Zymography TOP

• [01] What is zymography?
  Zymography is a method of detecting the activity of enzymes separated by electrophoresis. When a gel containing a substrate protein such as gelatin or casein is used, the entire gel is stained blue by protein staining, but enzymatically-active regions are detected as transparent bands due to degradation of substrate proteins.
• [02] Why can proMMP without enzyme activity be detected as a band by zymography?
  Since proMMP masks the active site with a propeptide sequence, it is inactive in vivo. In zymography, SDS treatment releases the propeptide sequence from its active site, thereby liberating its enzymatic activity.
• [03] How long does it take to perform a gel run with a constant current of 15mA?
  It depends on the device you are using, but, in general, it will take about 3 hours.
• [04] How do I take good quality gel pictures?
  In order to image gel bands clearly, we recommend white light illumination from below the gel. Although gel imaging devices are often combined with a UV transilluminator, a special "UV-white conversion screen" that converts UV into white light (UV/WL conversion screen) can be placed on the UV illuminator to create the optimal imaging environment. In addition, transilluminators equipped with white light along with UV, or transilluminators equipped with only white light are also available (UltraThin white LED panel through Cosmo Bio, TLW-20 Transilluminator and TMW-20 Transilluminator through BM Equipment). In addition to these approaches, digital cameras may also be used for image acquisition.
• [05] How much protein should I apply per lane?
  Since MMP activity in samples differs by tissue and cell type, it is difficult to predict the optimum amount of protein to be applied. We recommend a preliminary study to determine the optimum concentration based on user results, typically beginning with about 10μg protein. Also, when culture supernatant is used as a sample, it is often based on the amount of liquid.
• [06] How do I extract MMPs from tissues?
  MMPs are tightly bound to matrix components, so in general extraction efficiency from tissues is poor. The extraction method is to homogenize the tissue with a polytron in a buffer containing 10 mM CaCl2 and 0.25% Triton X-100 and collect the supernatant by centrifugation. Use the supernatant as a sample. Extraction methods from other tissues include solubilization with urea and heat treatment at 60°C. For details, see Woessner Jr's paper (Methods in Enzymology Vol.248, pp510-529).
• [07] What should I avoid during sample processing?
  When a reducing agent such as 2-mercaptoethanol or dithiothreitol (DTT) is added to the sample or when it is heat-treated, the protein is denatured and enzyme activity will be inactivated.
• [08] What are the effects of anticoagulants used in blood collection?
  In a paper by Gerlach et al. (Analytical Biochemistry Vol.344, pp147-149), there is a comparison of the effects of three anticoagulants (citric acid, heparin, and EDTA). Please see the paper for details.
• [09] What are the sizes of the precast gels included in the Gelatin Casein Zymo Electrophoresis Kit?
  Click here for a precast gel plate scale illustration

Gelatin Zymography TOP

• [01] What is the concentration of the precast gels?
  They have an acrylamide concentration of 10% and contain 0.1% gelatin.
• [02] What is the band detected between proMMP2 and MMP2?
  It is an intermediate that arises during MMP2 activation when the propeptide moiety is cleaved from proMMP2.
• [03] Is the mobility of proMMP9 in mice and humans different?
  MMP9 has N-type and O-type sugar chains, and the size of the sugar chains that bind to them differs depending on the animal species. Therefore, mouse-derived substances are detected on the polymer side compared to human-derived substances.
• [04] What are the bands detected on the polymer side of proMMP9?
  It is possible that these include MMP9 dimers and other complexes as well as other proteins (TIMP, lipocalin, α2-macroglobulin, etc.).

Casein Zymography TOP

• [01] What is the concentration of the precast gels?
  They are gradient gels with an acrylamide concentration of 8 to 13% and contain 0.05% casein.

Citations TOP

1. 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) (PMID: 21337372)
2. Fujiwara, M., 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. 32, 611-622 (2011) (PMID: 21360024)
3. 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) (PMID: 18309148)
4. 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 Transplanted Adipocyte Survival in Mice.
   Eur. J. Clin. Invest. 38, 752-759 (2008) (PMID: 18837800)
5. Fujimoto, M., 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) (PMID: 18560439)
6. Nakano, K., Hokamura, K., Taniguchi, N., Wada, K., Kudo, C., Nomura, R., Kojima, A., Naka, S., Muranaka, Y., Thura, M., Nakajima, A., Masuda, K., Nakagawa, I., Speziale, P., Shimada, N., Amano, A., Kamisaki, Y., Tanaka, T., Umemura, K., Ooshima, T.
   The collagen-binding protein of Streptococcus mutans is involved in haemorrhagic stroke.
   Nat. Commun. 2, 485 (2011) (PMID: 21952219)
7. Tahara, S., Nojima, S., Ohshima, K., Hori, Y., Kurashige, M., Wada, N., Ikeda, J., Morii, E.
   S100A4 accelerates the proliferation and invasion of endometrioid carcinoma and is associated with the "MELF" pattern.
   Cancer Sci. 107, 1345-52 (2016) (PMID: 27348205)
8. Lourbakos, A., Yau, N., de Bruijn, P., Hiller, M., Kozaczynska, K., Jean-Baptiste, R., Reza, M., Wolterbeek, R., Koeks, Z., Ayoglu, B., de Klerk, D., Campion, G., Zaharieva, I., Nadarajah, VD., Nilsson, P., Al-Khalili Szigyarto, C., Muntoni, F., Lochmuller, H., Verschuuren, JJ., Goemans, N., Tulinius, M., Niks, EH., de Kimpe, S., Aartsma-Rus, A., 't Hoen, PAC., Spitali, P.
   Evaluation of serum MMP-9 as predictive biomarker for antisense therapy in Duchenne.
   Sci. Rep. 7, 17888 (2017) (PMID: 29263366)