April 7th, 2015
SDS-PAGE Preparation
Solutions were prepared for SDS-PAGE analysis (to be run tomorrow).
Solutions:
- 50 mM Glycine-HCl, pH 3: prepared by dissolving 0.380 g of glycine in 100 mL of water with the addition of 3 M HCl dropwise to achieve the desired pH
- Running buffer (prepared previously-see 02/04/2015)
The protocol for SDS-PAGE was found here. The BioRad protocol can be found here.
Sample Prep:
- 10 µL of each original reaction sample was diluted to 1 mL with Gly-HCl buffer
- The sample from the last timepoint was used for each protease and at each concentration (1 µM, 100 nM, 10 nM, 1 nM)
- 10 µL of the glycine-diluted sample was further diluted with 10 µL of SDS-PAGE running buffer
- In order to account for potentially low concentrations of free protein in samples, a separate set of epitubes were prepared containing just 10 µL of the original reaction sample in addition to 10 µL of the running buffer, skipping the dilution step
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