Gelatin-Zymography Kit


print

Background

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.

Feature and Advantages

The size of the glass plate gel is 100(W)×100(H) and the thickness is 1mm.
One kit contains regents for 60 samples.

 

The precast gel which a component of this kit compatible with the following;

Cosmo Bio Electrophoresis Series: i-MyRun.P
Life technologies: Novex®
BIO CRAFT: Electrophoresis System
etc...

Kit component

ComponentQuantityStorage
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

< Protocol >

1. Load 100-150 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.

2. Take out the comb on precast gel carefully and set the gel to the electrophoresis chamber. The side of the sample holes should be set on the upper side. If the sample holes are disturbed, fix them up with a needle etc.

3. Load approximately 100 mL of Electrophoresis Buffer to the upper chamber (cathode).

4. Mix samples with equivalent volume of sample preparation buffer. Incubate for 15 minutes at Room Temperature. (Do NOT heat the samples.)

5. Load approximately 100 mL of Electrophoresis Buffer to the upper chamber (cathode).

6. Apply the samples and MMP markers to gel plate. (Fig.1: 10ml 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)
*MMP markers can be used without the sample preparation buffer.

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 the gel plate and peel off the gel carefully with a spatula.

9. Put the gel in the tray with 200 mL of Washing Buffer. Incubate with shaking 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 37°C for 20-40 hrs (Lower enzyme concentration needs longer reaction time.).

11.  After the 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 30minutes – several hours to de-stain.

 

< Application Examples >


Fig.1  10ul of MMP markers applied and enzymatic reacted for 24 hrs. at 37°C

 

< References >
(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)
(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.
(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)
(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)
(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)

Product List

Product Name Cat# Quantity Price

Gelatin Zymography Kit (Cosmo Bio type) DataSheet ,

PMC-AK47-COS

1 KIT ¥68,400
$912

Precast Gelatin Zymogram Gel (Cosmo Bio type) ,

PMC-AK48-COS

5 PLATE ¥23,400
$312

Precast Gelatin Zymogram Gel (Cosmo Bio type) ,

PMC-AK50-COS

10 PLATE ¥45,600
$608

MMP Marker DataSheet ,

PMC-AK38-COS

200 UL ¥23,000
$307

Gelatin zymography kit (ATTO type) DataSheet ,

PMC-AK45-COS

1 KIT ¥68,400
$912

Precast Gelatin Zymogram Gel (ATTO type) ,

PMC-AK46-COS

5 PLATE ¥23,400
$312

Precast Gelatin Zymogram Gel (ATTO type) ,

PMC-AK49-COS

10 PLATE ¥45,600
$608

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



“Gelatin-Zymography Kit” also belongs to the following categories.