User:Alexander Cvitan/Notebook/Experimental Biological Chemistry Lab/2013/09/25

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Objective

  • Complete the procedure outlined by Dr.Hartings here.
  • Run SDS-PAGE samples that were prepared yesterday and run densitometry of the bands to determine the amount of protein present.

Procedure

  • The gel was loaded as follows,<br.>
  • Well 1. 100 times diluted hemoglobin and dye<br.>
  • Well 2. Pepsin Time 1<br.>
  • Well 3. Pepsin Time 2<br.>
  • Well 4. Pepsin Time 3<br.>
  • Well 5. Pepsin Time 4<br.>
  • Well 6. Pepstatin Time 1<br.>
  • Well 7. Pepstatin Time 2<br.>
  • Well 8. Pepstatin Time 3<br.>
  • Well 9. Pepstatin Time 4<br.>
  • Well 10. 100 Times diluted hemoglobin and dye<br.>
  • Well 11. Dr.Fox's Sample<br.>
  • Well 12. 100 times diluted hemoglobin and pepsin<br.>
    • Note that a mistake was made while loading, the gel doesn't have a standard for 100x diluted hemoglobin with pepstatin. <br.>
  • Also Note that the overnight pepsin and pepstatin samples were viewed using UV-Vis today, as outlined in the procedure. Data is presented; however, in yesterdays writeup.

Data

  • Please note that some of the contents of well 11 leaked into well 10.
  • Wells are labeled left to right.

Sept 25 gel.png

  • Based off of these results it becomes apparent that the pepsin most likely wasn't active. Looking at the bands their really isn't any visual difference in band intensity except for the standard. This test should be run again with another sample of pepsin to ensure that that was the problem.<br.>