Zymogram Protocol
(for MMP2 and MMP9 detection)

Materials
  1. Mammalian cells
  2. Serum-free media (SFM): DMEM w/ or w/o 1% 100X penicillin+streptomycin
  3. Regular growth media: DMEM + 10% FBS + 1% 100X penicillin+streptomycin
  4. Sterile PBS
  5. 6-well plate
  6. Gel apparatus and running buffer
  7. 10% Zymogram gel (Invitrogen: EC6175BOX)
  8. 4x Sample Buffer without BME (4XSBw/oBME)
  9. Prestained protein molecular weight markers (Pierce Prod# 26681)
  10. Solution 1A:
    11. Concentration Ingredients (1L)
    50mM Tris-Cl, pH7.5 6.06 g Tris
    3 mM Azide 0.195 g Sodium Azide (or 3ml of 1M stock solution)
    2.5% Triton X-100 25 ml Triton X-100
    pH7.5 ~ 29 ml 1M HCl
    Dissolve Ingredients in about 800 ml H2O, adjust pH to 7.5 with 1M HCl as needed, add H2O to 1L. Filter (0.22 uM). Store at room temperature.
  11. Solution 2A:
    12. Concentration Ingredients (1L)
    50mM Tris-Cl, pH7.5 6.06 g Tris
    3 mM Azide 0.195 g Sodium Azide
    2.5% Triton X-100 25 ml Triton X-100
    5 mM CaCl2 5 ml 1M CaCl2
    1 uM ZnCl2 1 ml of 1 mM ZnCl2
    pH7.5 ~ 29 ml 1M HCl
    Dissolve Ingredients in about 800 ml H2O, adjust pH to 7.5 with 1M HCl as needed, add H2O to 1L. Filter (0.22 uM). Store at room temperature.
  12. Solution 3A:
    13. Concentration Ingredients (1L)
    50mM Tris-Cl, pH7.5 6.06 g Tris
    3 mM Azide 0.195 g Sodium Azide
    5 mM CaCl2 5 ml 1M CaCl2
    1 uM ZnCl2 1 ml of 1 mM ZnCl2
    pH7.5 ~ 29 ml 1M HCl
    Dissolve Ingredients in about 800 ml H2O, adjust pH to 7.5 with 1M HCl as needed, add H2O to 1L. Filter (0.22 uM). Store at room temperature.
  13. Coomassie Blue solution:
    14. Concerntration
    Ingredients (1L)
    0.2% Coomassie Blue 2g Coomassie Blue(R-250)
    20% Methanol 200 ml Methanol
    7.5% Acetic acid 75 ml Glacial acitic acid
    Dissolve ingredients in MilliQ H2O and fill up to 1L, filter. Store at RT.
  14. Destain solution: 20% acetic acid, 5% methanol in H2O
  15. Container (pan) for wash, stain and destain.
  16. 37oC incubator
  17. Rotator
Procedure
  1. Seed cells at 1 x 106/well for eppithilium cells or 5 x 105/well for fibroblast cells in regular growth media in 6-well plate and incubate at 37oC with 5% CO2 for one day.
  2. When the cells are almost 100% confluent, wash cells twice with SFM and add 800 (-1000) ul SFM/well. Continue incubation at above condition for 24-48 hours.
  3. Collect the conditioned media into labeled Ependorf tubes, and place on ice.
  4. Spin tubes in centrifuge at 4oC to separate cells. Transfer the supernatents into fresh tubes.
  5. (The sample can be stored at -80oC at this step. )
  6. Pipette 24ul of the media from above into another Ependorf tube and add 8 ul of 4XSBw/oBME to each tube and vortex to mix well. Spin briefly to collect all liquid at bottom of tube.(NO HEATING!)
  7. Also at this time reconstitute the protein molecular weight marker mix with 10 ul MilliQ H2O and mix with 20 ul 1X SB so you can load the same volume as of samples.
  8. Assemble Xcell SureLock Mini-Cell:

    1) Lower Buffer core into the lower buffer chamber so that the negative electrode fits into the opening in the gold plate.

    2) Insert gel tension wedge behind the buffer core. Make sure the wedge is in its unlocked position. It should rest on the bottom of the lower buffer chamber.

    3) Remove gel cassette from pouch by cutting with scissors, drain packing buffer, rinse with deionized water.

    4) Peel off tape covering slot on back of gel cassette.

    5) In one fluid motion, pull comb out of cassette.

    6) Insert gel cassettes into the lower buffer chamber. Place one cassette behind the core and one cassette in front of the core. For each cassette, the shorter "well" side of the cassette faces in towards the buffer core. The slot on the back of the cassette must face out towards the lower buffer chamber.

    7) If you are running only one gel, replace the rear gel cassette with the buffer dam.

    8) Pull forward on the gel tension lever in a direction towards the front of the Xcell SureLock unit until lever comes to a firm stop and the gels appear snug against the buffer core.

    9) Fill the upper buffer chamber (between the two gels) to the top of the gel cassettes with 1X Running Buffer. Ensure that the chamber is not leaking by observing level. If the level of running buffer drops, the electrophoresis core and cassettes are not properly seated.

    Fill the lower buffer chamber with 200 ml of the 1X running buffer.

    10) Use a loading pipette to gently wash the cassette wells with the running buffer in the upper chamber.

  9. Apply samples (typically 28 ul) into the wells by lowering the pipette tip to the bottom of the sample well and slowly pipetting sample into well without contaminating another well with sample. In lane 1 load the the molecular weight marker, and in lanes that do not have sample, load the same volume of 1X SB.
  10. Run the gel according to the following running conditions:

    Voltage: 200 V constant

    Run Time: Approximately 60 minutes. The run is complete when the bromophenol blue tracking dye reaches the bottom of the gel.

  11. After running, remove the lid and unlock the gel tension lever. Remove the cassettes from the mini-cell by handling edges only. Remove gel carefully from cassette.
  12. Place gel in Solution 1A for 20 minutes with gentle agitation. Rinse with deionized water for 5 minutes. Repeat with Solutions 2A and 3A. When done with 3A, replace solution with fresh 3A.
  13. Place lid on the pan and incubate at 37oC for 24 hours.
  14. Wash with d H2O for 5 min.
  15. Stain with Coomassie Blue for one hour.
  16. Destain for about an hour. Areas of protease activity will show up as clear bands.
  17. Store gels in water until photograph and package.
  18. Package gel with pouche. When seal, carefully remove all bubbles. The gel can be kept at room temperature.

 
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