SDS-PAGE Protein Gels: Difference between revisions
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==='''''For 5GB1 membrane-separated protein SDS-PAGE'''''=== | ==='''''For 5GB1 membrane-separated protein SDS-PAGE'''''=== | ||
=='''Considerations for Loading the Gel'''== | =='''Considerations for Loading the Gel'''== |
Revision as of 15:49, 20 May 2013
Consolidated/Pictorial Protocol
Sample Prep - Marina Protocol
For 5GB1 whole cell protein SDS-PAGE
- 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
At this point, pellet can be frozen and the following steps can be performed at a later date
- Mix Laemmli sample buffer using the following recipe:
- 500 ul Bio-Rad 4x Laemmli buffer
- 450 ul dH2O
- 50 ul 2-mercaptoethanol
- Add 100 ul of buffer to pellet and vortex to ensure cells and Laemmli buffer are well mixed
- Incubate sample for 5 minutes at 95-100°C
Samples are ready to be loaded into gel as described below
For 5GB1 membrane-separated protein SDS-PAGE
Considerations for Loading the Gel
- Note that gels are both concentration- and volume-dependent; overloading of either will cause deformation of lanes and smearing
- 15 ul of 5GB1 prepared as above gives pretty good banding and a concentration ~4ug/ul
- Use 5 ul ladder
- Use BSA (usually comes at [10 ug/ul]) to make standards for calibration curve
Running the Gel
- Bio-Rad Mini-Cell Setup
- Use diluted 10X Tris/Glycine/SDS Buffer
- Re-using running buffer 2-3 times does not affect results
- If only running one gel, don't forget to 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
- Pour enough to 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!
- 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.