SDS-PAGE Protein Gels
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(New page: === Consolidated/Pictorial Protocol === SDS casting protocol =='''Sample Prep - Marina Protocol'''== '''''For 5GB1 SDS-PA...)
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Revision as of 17:55, 20 May 2013
Sample Prep - Marina Protocol
For 5GB1 SDS-PAGE total protein concentration estimation
- Grow cells to ~ OD 1.0
- For calculation purposes, you ideally want 1 ml of cells at an OD of 1.0
- If your samples are not at 1.0, increase or decrease volume to compensate (eg: If OD is only 0.747, use 1.34 ml)
- Weigh eppindorf tube
- Need to subtract tube weight to determine wet cell weight (WCW) of sample -- don't forget this step!
- Harvest appropriate sample volume and pellet cells
- RT benchtop centrifugation at 14K RPM; 2-3 minutes should be more than sufficient
- Dry the pellet as completely as possible by decanting and then pipetting off any remaining liquid
- Weigh dried pellet and eppindorf tube, subtract out tube weight and record WCW
Running the Gel
- Bio-Rad Mini-Cell Setup
- If only 1 gel, use buffer dam to replace second gel
- Position the gels with the shorter plate facing inward!
- Apply pressure on gel holder and gels as you close the tabs to seal the center compartment.
- Fill central compartment with running buffer
- should fill sample wells
- Pour more into the outer compartment to specified line
- Load gel
- Make sure you will be able to determine the orientation of your gel after it is stained. Asymmetry is good!
- Make sure to color/charge-match the cords to the power unit as the electrodes in the gel holder to the contacts in the lid.
- Run @ 60 V for ~15 min, then 200 V for ~ 20+ min.
- Note: ladder looks blurry while running through the stacking gel; don't be alarmed unless it still looks blurry in the resolving gel.
- Can skip the 60V step if you don't need a gorgeous gel
- Amanda runs 20 min at 200V, then checks frequently to make sure the protein doesn't run off the gel.
Storing Samples After Use
- Amanda was taught to flash-freeze (liquid N2) samples and store them at -80oC. Janet loves liquid N2 (particularly dumping it on the ground after use) so I follow along without question.
- Ladder is stored at -80oC. An aliquot of PageRuler Prestained that sat out at room temp for 1 week looked perfect in a gel, so don't worry about that ladder's stability.