One-dimensional SDS-PAGE (9-18% TGX gel)
Sigrid Verhelst
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Abstract
Protocol for one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 9-18% TGX gel for visualization and quantification of histone proteins.
Steps
Sample preparation
Dry samples (equal to 400.000 cells)
Resuspend samples in 10µl laemmli-buffer
Add 1µl β-mercaptoethanol to each sample
Vortex and spin down
Incubate for 7 minutes at 95°C in a thermoshaker
Spin down
Prepare Criterion Cell
Place the criterion cell on ice in a fume hood
Remove the sticker from the bottom of the gel cassette and check the gel for cracks
Put the gel cassette in the criterion cell
Fill the reservoir with running buffer (25mM Tris, 0.1% SDS, and 192mM glycine in MilliQ water) and take out the comb
Running of the samples
Load the samples and standards (2 µg of bovine histones) on the gel (3 standards per gel: lane 1, lane 9 and lane 18)
Put the cover on the criterion cell
Start running the gel on 200V
Stop running when the frontline is almost gone
Visualization
Take out the cassette
Incubate in fixation-solution (7% acetic acid, 10% methanol in MilliQ water) for 10 minutes on a shaker
Wash the gel 3 times for 5 minutes in MilliQ water on a shaker
Incubate in SyproRuby overnight
Wash the gel 3x for 10 minutes in MilliQ water on a shaker
Visualize the gel (Versadoc)
