Difference between revisions of "Team:UCLA/Notebook/Protein Cages/20 August 2015"

Latest revision as of 02:14, 21 August 2015

iGEM UCLA

Phillip's notes:

Introduction: Today, DLS will be done on the purified PCquadY from last week. The elutions will be pooled before bringing it to Kevin.

Procedure: The first two elutions from the experiment visibly contained a white precipitate, which is likely the protein cage. These tubes were pooled and centrifuged. The pellet was re-suspended in 150uL of triton lysis buffer.

Results: The supernatant sample and the re-suspended pellet were both ran. Both did not provide a baseline reading.

Conclusion: Since aggregation seems to be a problem, the DLS may have to be ran soon after purification.

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 Phillip's notes:
-Introduction:  BCA assay will be done on the final elution of the previous affinity purification.  According to Kevin from the Yeate’s lab, a minimum of 2mg/mL of protein is needed for the DLS assay.
-Procedures:
+Introduction:  Today, DLS will be done on the purified PCquadY from last week.  The elutions will be pooled before bringing it to Kevin.
-The standard procedure was followed in the Thermo BCA kit in order to prepare the standard solutions.  All standards were diluted in triton lysis buffer.
-Elution 3 was used because it appeared to be the purest.  Upon starting the assay, it was noted that some of the protein was aggregating and precipitating out of solution.  The tube was spun down at max speed for 5 minutes, and the supernatant collected.  The samples ran were the following:
+Procedure:
+The first two elutions from the experiment visibly contained a white precipitate, which is likely the protein cage.  These tubes were pooled and centrifuged.  The pellet was re-suspended in 150uL of triton lysis buffer.
-Elution with aggregate, supernatant, 1:10 supernatant, and 1:100 supernatant.
+Results:  The supernatant sample and the re-suspended pellet were both ran.  Both did not provide a baseline reading.
-A standard curve was constructed on excel.  Only absorbance readings at about .1 to 1 were used.  Only the original elution showed a measured protein concentration at about 620ug/mL.
+Conclusion:  Since aggregation seems to be a problem, the DLS may have to be ran soon after purification.
-Conclusion:  It appears that most of the protein cage is aggregating out of solution.  Perhaps this can be used in order to separate the protein cage in the other two elutions, and resuspend them in a higher volume.