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FITC-labeled Gelatin-zymography Kit

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 FITC-labeled Gelatin-Zymography Kit (Cat.No.PMC-AK83-COS) 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 fl uid, secretion, cell lysate, cell culture medium, and other samples.

Features

• Precast gel and reagents for zymography are included in this kit.
• You can check the enzyme reaction continuously.

Kit Components

Description Quantity
Precast gel for 12 samples 5 pieces
Electrophoresis Buffer ( × 10) 100 mL x 2
Washing Buffer ( × 10) 100 mL x 1
Reaction Buffer ( × 10) 25 mL x 1
Sample Preparation Buffer 5 mL x 1
MMP markers (ProMMP-2, MMP-2, ProMMP-9) 0.2 mL x 1

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

Preparation of reagents

• Electrophoresis Buffer
  Dilute Electrophoresis Buff er ( × 10) with distilled water to a 1x concentrated solution.
Note: Diluted buff er should be stored at 4°C.

• Washing Buffer
  Dilute Washing Buff er ( × 10) with distilled water to a 1x concentrated solution.
Note: Diluted buff er should be stored at 4°C.

• Reaction Buffer
  Dilute Reaction Buff er ( × 10) with distilled water to a 1x concentrated solution.
Note: Diluted buff er should be stored at 4°C.

Preparation of samples

Mix Sample Preparation Buff er and a samples in the ratio of 1 to 1 and leave it in room temperature for 15 minutes.
Note: Do not heat the samples.

Protocol

Prepare tubes for sample, blanks, and Substrate Solution.

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 buff er.

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 are disturbed, fi x them up with needle etc.

3. Load around 100 mL of Electrophoresis Buff er to the upper chamber (cathode).

4. Apply samples and MMP markers to gel plate. (Fig.1: 10 µL of MMP markers applied and enzymaticreacted for 24 hrs at 37℃ ).

5. Run electrophoresis at 15 mA constant current. (If you use 2 gels, set the current at 30 mA)
*MMP markers can be used without sample preparation buff er

6. After the run is completed, turn off the electrophoresis chamber and take out the gel plate from the chamber.

7. Remove the upper glass plate and peel off the gel from the lower glass plate (the biggest glass plate) carefully with a spatula.

8. Put the gel in a tray with 200 mL of Washing Buff er. Incubate with shaking at Room Temperature.

9. Put the gel in a container with 50 mL of Reaction Buff er and seal up the container. Incubate the gel at 37℃ for 20-40 hrs. (Lower enzyme concentration needs longer reaction time.)

10.After enzymatic reaction, remove the gel and take a photo by gel imaging system. (fluorescent wavelength: 535 nm)

Electrophoresis patterns

Fig. 1 Electrophoresis pattern of MMP marker

Fig. 2 Electrophoresis pattern for human tear samples
① Molecular-weight marker
② MMP marker
③~⑧ human tear samples (6 lanes)

Product List

Product Name Cat# Quantity Price

FITC-labeled Gelatin-zymography Kit (Cosmo Bio type)

PMC-AK83-COS 1KIT

¥ 75,000
$ 1000
€ 750

FITC-labeled Gelatin-zymography Precast Gel (Cosmo Bio type)

PMC-AK84-COS 5PLATE

¥ 35,000
$ 467
€ 350

FITC-labeled Gelatin-zymography Precast Gel (Cosmo Bio type)

PMC-AK85-COS 10PLATE

¥ 65,000
$ 867
€ 650

MMP Marker

PMC-AK38-COS 200UL

¥ 23,000
$ 307
€ 230

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