Jan 25, 2024
One-dimensional SDS-PAGE (9-18% TGX gel)
- Sigrid Verhelst1
- 1ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium
Protocol Citation: Sigrid Verhelst 2024. One-dimensional SDS-PAGE (9-18% TGX gel). protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pz27g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2024
Last Modified: January 25, 2024
Protocol Integer ID: 94076
Keywords: dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresi, gel electrophoresi, quantification of histone protein, histone protein, electrophoresi, histone, protein
Funders Acknowledgements:
FWO
Grant ID: 3S031319
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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.
Troubleshooting
Sample preparation
Dry samples (equal to 400.000 cells)
Resuspend samples in 10µl laemmli-buffer
Add 1µl β-mercaptoethanol to each sample
Safety information
Perform this step in a fume hood
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
Overnight
Wash the gel 3x for 10 minutes in MilliQ water on a shaker
Visualize the gel (Versadoc)