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Gelatin Zymography
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Materials Required:

 

  • Gelatin solution
  • Acrylamide solutions (for resolving & stacking gels)
  • 1.5M Tris
  • 1M Tris
  • 10% SDS
  •  Water saturated butanol/distilled water.
  • 4X sample buffer
  • Running buffer
  • Protein samples
  • Triton X 100
  • Trichloroacetic acid [TCA]
  •  Low salt collagenase buffer
  • Staining and destaining solutions

 

The equipment and supplies necessary for conducting Zymography include:


    1)    An electrophoresis chamber and power supply
    2)    Glass plates(a short and a top plate)
    3)    Casting frame
    4)    Casting stand
    5)    Combs

 

Composition of Resolving gel [10%]:

 

 

Composition of Stacking gel [5%]:

 

 

Caution:

 

  1. Gloves should be worn at all times while performing Zymography.
  2. To insure proper alignment and casting, the glass plates, combs and casting stand gaskets must be clean and dry.
  3. Special attention should be paid while using acrylamide (since it is a neurotoxin).

 

      I)   Assembling the glass plates

 

  1. Assemble the glass plate on a clean surface. Lay the longer glass plate (the one with spacer) down first,then place the shorter glass plate on top of it.
  2. Embed them into the casting frame and clamp them properly make sure that the bottom ends of the glass plates are properly aligned.
  3. If so place it on the casting stand.

 

      II)  Casting the gels

 

        Prepare 10% Resolving/separating gel and 5% stacking gel using the  composition given in the section 3 .

 

        Prepare the resolving gel solution by combining all reagents including gelatin except ammonium persulfate (APS) and TEMED.

 

  1. Add APS and TEMED to the monomer solution (just before pouring ) and mix well by swirling gently. Pour the solution till the mark.(It is ok if you introduce air bubbles, add a layer of water saturated butanol or distilled water on top of the gel so as to level the poured gel).
  2. Allow the gel to polymerize for 20-30 minutes.
  3. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Drain the water saturated butanol  with  strips of filter paper.
  4. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently.(Make sure you keep the comb ready by the side).
  5.  Immediately place a comb in the stacking gel sandwich. Allow it to polymerize for at least 20 minutes.

 

     III) Preparation of samples


          Mix your protein in the ratio of 4:1 with the sample buffer.


     IV)  Running the gel:

 

  1. To assemble, take out the gels from the casting frame and clamp them in the gel apparatus.( Make sure that the short plate always   faces   inside and if you have got only one gel to run use the dummy plate that is available to balance).
  2. When the plates are secured, place them in the cassette and lock it.
  3. Place them in the tank.
  4. Fill the inner chamber of the tank with buffer. (Now it is easy to remove the comb, since it is lubricated).
  5. Remove the comb carefully (without breaking the well). [Now the gel is ready to load the samples].
  6. Rinse the loading tip a few times with running buffer. (Make sure that all the buffer  is poured out before loading the samples).
  7. Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse the syringe a few times with distilled water after loading.
  8. Attach the power supply by putting the lid (Make sure that the connection is correct,ie.,Black lead to black and red lead to red). Set the voltage up to 150 V and run for 1 hour till the dye front reaches the end of the gel.
  9. Take out the gel and put it in 50 ml of triton X 100 (5%) and incubate for 5 minutes in an orbit shaker. Repeat this step thrice.
  10. Discard the triton X 100 solution and add about 100ml of  low salt collagenase buffer to the gel and keep in the orbit shaker for 5mintes . Repeat this step thrice.
  11. Add 100 ml low salt collagenase buffer to the gel and keep for minimum 4 hours to overnight incubation in an orbit shaker.
  12. Next day, discard the low salt  collagenase buffer  and pour Trichloroacetic acid to the gel and incubate for 30 minutes in an orbit shaker.
  13. Discard the TCA solution and add staining solution and keep for half an hour incubation in an orbit shaker.
  14. After 30 minutes in incubation with staining solution, pour destaining solution and keep until the clear bands are visible against a   blue     background. Incubation time needs to be optimized depending on the enzyme activity. Excess incubation time might obscure the sensitivity for quantitation.

 

 

 

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