User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/04/07

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

April 7th, 2015

SDS-PAGE Preparation

Solutions were prepared for SDS-PAGE analysis (to be run tomorrow).

Solutions:

  1. 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
  2. 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



Personal tools