SDS-PAGE Protein Gels: Difference between revisions
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'''''At this point, pellet can be frozen and the following steps can be performed at a later date''''' | '''''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: | *Mix Laemmli sample buffer using the following recipe: | ||
::500 ul | ::500 ul Bio-Rad 4x Laemmli buffer | ||
::450 ul dH2O | ::450 ul dH2O | ||
::50 ul 2-mercaptoethanol | ::50 ul 2-mercaptoethanol | ||
Line 19: | Line 19: | ||
*Incubate sample for 5 minutes at 95-100°C | *Incubate sample for 5 minutes at 95-100°C | ||
'''''Samples are ready to be loaded into gel as described below''''' | '''''Samples are ready to be loaded into gel as described below''''' | ||
'''''As of May 2013 5GB1 proteins are analyzed using Bio-Rad Mini-Protean TGX Precast Gels, 12% polyacrylamide (Cat# 456-1045)''''' | |||
Revision as of 15:15, 20 May 2013
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 As of May 2013 5GB1 proteins are analyzed using Bio-Rad Mini-Protean TGX Precast Gels, 12% polyacrylamide (Cat# 456-1045)
Consolidated/Pictorial Protocol
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.