User:Roberta Diaz Jimenez/Notebook/CHEM 471 Experimental Lab/2015/10/07: Difference between revisions

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==Procedure==
==Procedure==
In order to do the Bradford Assay of Protease Degradation, we followed Dr. Hartings' [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2015/09/29|protocol]], and did the following specific steps:
*Protease Sample Prep.
*Protease Sample Prep.
#Used eppendorf tube no. 11 that weighed 1.02097g, and contained 0.00154g.
#Used eppendorf tube no. 11 that weighed 1.02097g, and contained 0.00154g.
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##0.985mL of  tris buffer
##0.985mL of  tris buffer
##0.015mL of trypsin solution
##0.015mL of trypsin solution
*Additional specifications:
#Prior to prepping the samples, the eppendorf tubes containing the fibers were centrifuged for 10 mins at 300rpm.
#After heating the samples, they were all centrifuged for 1 min. at 13'200rpm. This was done only for the samples, not the blanks.
#The Bradford dilution was 1:4; with 1mL of Bradford and 3mL of buffer.


==Data==
==Data==

Revision as of 10:28, 13 October 2015

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Objective

Measure protease Trypsin kinetics with Bradford Assay using 1µM of trypsin.

Procedure

In order to do the Bradford Assay of Protease Degradation, we followed Dr. Hartings' protocol, and did the following specific steps:

  • Protease Sample Prep.
  1. Used eppendorf tube no. 11 that weighed 1.02097g, and contained 0.00154g.
  2. Added 1mL of phosphate/CaCl2 buffer.
  3. Final concentration: 66.09µM
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers.
  2. To each tube add:
    1. 0.985mL of buffer
    2. 0.015mL of trypsin (add this at the time of putting the tubes in the 37˚C hot water bath).
  • Blank Prep.
  1. In on eppendorf tube add:
    1. 0.985mL of tris buffer
    2. 0.015mL of trypsin solution
  • Additional specifications:
  1. Prior to prepping the samples, the eppendorf tubes containing the fibers were centrifuged for 10 mins at 300rpm.
  2. After heating the samples, they were all centrifuged for 1 min. at 13'200rpm. This was done only for the samples, not the blanks.
  3. The Bradford dilution was 1:4; with 1mL of Bradford and 3mL of buffer.

Data


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