Exercise 6.3 Gelatin Zymography

Exercises

Exercise 6.3 Gelatin Zymography

  1.  Obtain 0.5mL of treated HL-60 cells from your TA.
  2. Centrifuge cells for 2min at 13.5rpm.
  3. Take 50µl of supernatant and place into fresh microfuge tube
  4. Add 50µl of 2X Tris-Glycine Sample Buffer to your tube and vortex.
  5. Let stand at room temperature for 10 minutes.
  6. Use precast gels purchased from Invitrogen.
  7. Load 25µl of your sample into the gel and run the gel with 1x Tris-Glycine SDS Running Buffer according to the standard running conditions (~125V, constant voltage).
  8. After running, dilute the Zymogram Renaturing Buffer (10x) 1:9 with deionized water and incubate the gel in the buffer (100 ml for one or two mini-gels) with gentle agitation for 30 minutes at room temperature.
  9. Decant the Zymogram Renaturing Buffer and replace with 1x Zymogram Developing Buffer (100 ml for one or two mini-gels).
  10.  Equilibrate the gel for 30 minutes at room temperature with gentle agitation then replace with fresh 1x Zymogram Developing Buffer and incubate at 37°C overnight for maximum sensitivity. Stain with Coomassie Blue R-250 for at least 1 hour.
  11. Gels should be destained with an appropriate Coomassie R-250 destaining solution (Methanol : Acetic acid : Water (50 : 10 : 40)). Areas of protease activity will appear as clear bands against a dark blue background where the protease has digested the substrate.
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Analyze the example Vision document provided as an attachment in this week’s module. Answer the following questions and submit as a Word document. Please provide thorough answers in complete sentences, demonstrating your understanding of the purpose of the document.

1. Overall, do you believe the HACS Vision document is well written and accomplishes the goals of this type of deliverable? Why or why not?

2. Is the Problem Statement structured in an easy to understand manner? Are any topics missing in your opinion? Give your opinion on Problem Statements presented in a table versus paragraphs of text.

3. Do the Stakeholder Profiles provide useful information? Are they redundant in any way?

4. The HACS Vision document lists 59 Product Features. How does this compare to the guidelines provided in Larman Chapter 7? If you were told to re-write the Product Features section, how could you structure it differently?

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