Team:UCLA/Notebook/Protein Cages/11 August 2015
Phillip's notes:
Introduction: An SDS-PAGE gel will be ran to analyze the results of yesterday’s purification.
Procedures:
All samples were obtained and put on ice.
Using 2x loading dye, 5% BME was added. 142.5uL of 2x dye to 7.5uL of BME.
15uL of each sample was mixed with 15uL of dye. 10uL of dual color ladder was mixed with 10uL of loading dye. Aggregation was observed in the whole cell lysate and in the elution fractions. All samples were boiled for 10 minutes, and centrifuged for 10 seconds to collect at the bottom. The aggregates were noticeably gone.
The SDS-PAGE apparatus was assembled, and 1L of running buffer was made from a 10x solution.
20uL of each sample was added to the wells. The gel was ran at 100V for 30 minutes to ensure even running, then at 120V until the dye front was near the bottom. ~1.5 hours.
The gel was cut out, and stained with coomassie for 1 hour. Destaining took place for 2 hours, with changing the destain buffer every hour.
Conclusions: Since the destain wasn’t completely done, the gel was left in destain on the bench overnight.
Results:
Below is the gel. From left to right: Ladder, ladder, whole cell lysate, unbound proteins, wash 1-3, elution 1-3.
Media:2015-08-12_SDSPAGE_Yeates_PCquad.jpg
It appears that a preliminary purification worked, though more washes can make it cleaner. To avoid the observed aggregation, more lysis buffer may be used next time.
Intro: Transformed Mutant # 10 into chemo-competent BL21-DE3 cells.
Transformation: