SDS-PAGE Protein Gels: Difference between revisions
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=== Consolidated/Pictorial Protocol === | |||
[[image:SDS-PAGE sample prep and loading.jpg|thumb|upright=3.0|center|SDS-PAGE sample prep and loading]] | |||
=='''Sample Prep - Marina Protocol'''== | =='''Sample Prep - Marina Protocol'''== | ||
'''''For 5GB1 whole cell protein SDS-PAGE''''' | '''''For 5GB1 whole cell protein SDS-PAGE''''' | ||
Line 19: | Line 21: | ||
*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)''''' | '''''As of May 2013 5GB1 proteins are analyzed using Bio-Rad Mini-Protean TGX Precast Gels, 12% polyacrylamide (Cat# 456-1045)''''' | ||
== | =='''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 '''== | =='''Running the Gel '''== | ||
*Bio-Rad Mini-Cell Setup | *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!''' | *'''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. | * Apply pressure on gel holder and gels as you close the tabs to seal the center compartment. | ||
[[image:gel holder.jpg|thumb|center|Mini-cell Gel holder]] | [[image:gel holder.jpg|thumb|center|Mini-cell Gel holder]] | ||
* Fill central compartment with running buffer | * Fill central compartment with running buffer | ||
:: Pour enough to fill sample wells! | |||
* Pour more into the outer compartment to specified line | * Pour more into the outer compartment to specified line | ||
* Load gel | * 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. | * 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== | ==Storing Samples After Use== | ||
* Amanda was taught to flash-freeze (liquid N<sub>2</sub>) samples and store them at -80<sup>o</sup>C. Janet loves liquid N2 (particularly dumping it on the ground after use) so I follow along without question. | * Amanda was taught to flash-freeze (liquid N<sub>2</sub>) samples and store them at -80<sup>o</sup>C. 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. | * 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. |
Revision as of 15:39, 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
As of May 2013 5GB1 proteins are analyzed using Bio-Rad Mini-Protean TGX Precast Gels, 12% polyacrylamide (Cat# 456-1045)
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.