Gelatinase spectrum MMP (2/9) detection experiment

Gelatinase spectrum MMP (2/9) detection experiment

Based on SDS-PAGE electrophoresis and reverse gel staining, zymography is used to detect the activity of MMP-2 and MMP-9, with a sensitivity of 10pg, higher than that of ELISA.

The basic principle and procedure are: use SDS-PAGE gel with collagenase substrate to separate the sample containing protease, take out the gel after electrophoresis and incubate it with enzyme reaction Bufer, and then dye and decolour the gel. The gel is deeply stained due to the presence of substrate protein, forming a dark background. However, where there is a protease band, the protease will degrade the substrate protein in the gel, so it will not be stained to form a transparent area, which can simultaneously indicate the position (zymogram) of MMP-2 and MMP-9, and can detect the presence of blood, exudate, cell extracts, etc.

Service cycle: 10 working days

Results provided: Results report, representative images, operating procedures, and relevant reagent and instrument information.

Gelatinase spectrum MMP (2/9) detection experimentSample provision requirements:

1) The customer informed us of the experimental grouping, numbering, and background design information. Please specify the specific requirements in advance;

2) Specimen collection, preservation, and transportation:

① Organizational samples: Fresh tissue should be stored in liquid nitrogen or at -70 degrees Celsius, with a minimum of 100mg of tissue;

② Cultivate cells: Take no less than 2x106 cells by cell counting and transfer them to an EP tube. Add 0.5ml of physiological saline, mix well, and store in a refrigerator (-70 ° C to avoid repeated freezing and thawing);

③ Blood cell specimen: Store the blood sample in an anticoagulant tube or separate the cells with lymphocyte separation solution and add 0.5ml of physiological saline for preservation (-70 ° C can be stored for a long time to avoid repeated freezing and thawing)

④ Serum or cell culture medium specimens: After centrifugation to remove impurities, take the supernatant and transfer it into an EP tube for storage (can be stored at 49C for 15-20 days, and can be stored for a long time at -70 ° C to avoid repeated freezing and thawing)

4) Sample transportation:

① Transport organizational samples and cell samples with dry ice;

② Cell samples can also be directly sent to culture bottles (sealed to ensure no contamination and no leakage of culture medium);

③ Serum or supernatant can be directly sent in ice packs (depending on the distance, it can take 2-3 days to arrive).

Customer Notice:

The detection system includes positive reference or internal reference, and the quotation includes protein extraction

Experimental precautions:

When preparing polyacrylamide, attention should be paid to eliminating bubbles.

2. The activity of gelatinase spectrum is affected by factors such as calcium ions, zinc ions, and pH value. Therefore, the preparation of buffer solution should be strictly accurate, ultrapure water should be used as much as possible, and the incubation temperature should be well controlled.

3. Use a large comb

4. The pH of the incubation solution should be between 7.5-7.6. After a long time of refolding TRITON, there will be flocculent substances, so fresh preparations should be used as much as possible during the experiment.

5. Do not incubate at 37 degrees Celsius in a CO2 incubator, as it will change the pH value of the incubation solution. It can be done in a regular incubator.

6. Gelatin should be stored at 4 degrees Celsius and used within one week after preparation

Experimental outsourcing service: