Lidstrom: SDS-PAGE: Difference between revisions
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== '''Recipes'''== | == '''Recipes'''== | ||
All recipes except the staining & destaining solution are from the [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10007296.PDF Mini-PROTEAN® Tetra Cell manual] | |||
=== Loading Buffer (5x): === | |||
** | ** You will mix pre-made 5X loading buffer with fresh beta-mercaptoethanol prior to each use. | ||
* | ** 5x loading buffer: | ||
* | * mix 50 uL beta-mercaptoethanol to 950 uL sample buffer prior to use. | ||
** Scaled back 10x: 10 uL beta-mercaptoethanol + 190 uL 5x buffer | |||
* Dilute the sample at least 1:2 with sample buffer and heat at 95oC for 4 min to lyse the cells. | |||
* Amanda mixes 160 uL 5X buffer, 8 uL beta-mercapto-ethanol, 32 uL H2O. Use 10 uL per 25 uL of cells. | |||
* ?? Should add up to 8M urea for really hydrophobic proteins | |||
* | === Running Buffer: === | ||
* 10x SDS running buffer (1 liter): 30.3 g Tris-HCl, 144 g Glycine, 10 g SDS. Fill to 1 L with ddH2O. Don't add acid or base to adjust pH. | |||
* | * Make 1L of 1x for use. | ||
*Store at 4oC. | |||
* This buffer is used while running proteins through the gel. Pour it in as the instructions for the box explain. Pour back into bottle for re-use afterward. | |||
* Keep until ____; remake after this. | |||
* Destaining Buffer: | === Staining Buffer(Coomassie Brilliant Blue G-250): === | ||
* 0.500 g Brilliant Blue, 500 mL methanol, 100 mL glacial acetic acid, 400 mL dH20. Mix well. Store at room temperature; can be reused 2-3 times. | |||
* note: Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two methyl groups. We use the G form. Read more about the R form [http://en.wikipedia.org/wiki/Coomassie_Brilliant_Blue here]. | |||
=== Destaining Buffer: === | |||
* | * | ||
Revision as of 13:43, 22 October 2012
Return: Protocols
Gel Prep
- Clean cover plate and thicker spacer plate 75 mM
- Soap and Water
- Ethanol
- DI water
- Dry plates
- Setup one spacer plate and one cover plate in each gel holder
- The cover plate goes on the side of the spacer plate with the spacers in order to create a small gap between the plates
- Put the gel holder into the casting stand
Pour the Resolving Gel
- Mix components of the amounts in the Gel Mix link for the resolving gel (Recipes is for 4 gels). Mix in the order listed.
- Gel Mix Recipe
- Don't add APS/TEMED until ready to pour
- The APS solution should be ~ 1-2 months old. A 3 month old solution failed to cause polymerization -JM 10/2012
- Use pipette to put gel mix into the gap between the plates
- Carefully layer 50%EtOH 50% ddH2O on top of the gel to prevent the top of the gel from drying out
- Let dry for an hour
- Store at 4 deg C wrapped in a wet paper towel and saran wrap if you're not going to use it right away.
Pour the Stacking Gel
- Replace gel in gel holder
- Dry surface of gel carefully with Kimwipe or paper towel
- It can be a little gooey
- Mix components of the amounts in the Gel Mix link. Mix in the order listed.
- Gel Mix Recipe
- Don't add APS/TEMED until ready to pour
- Use pipette to put gel mix into the gap between the plates
- Insert the comb being 'careful not to trap any bubbles'
- Attach binder clips to help hold the comb in while drying. One in either side of the casting stand clamp.
- Leave for 1 hour while polymerization occurs.
- Can store for a few weeks in the fridge. Leave comb in, and wrap in a wet paper towel and cling wrap.
Sample Prep
- 10-20 ug protein
- Load 12-15 uL, absolute max 20 uL for big comb
- Denature protein
- Mix with sample buffer & BME
- How much?
- Boil for 5-10 min (can do longer times at lower temp, too.)
- Mix with sample buffer & BME
Running the Gel
- Bio-Rad Mini-Cell Setup
- If only 1 gel, use buffer dam to replace second gel
- Slot gels with cover plates facing each other...
- Apply pressure on gel holder and gels as you close the tabs to seal the center compartment.
- Fill central compartment with running buffer
- Pour rest into outer compartment
- Load gel
- 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 30-45 min @250V
- Amanda runs 20 min at 200V, then checks frequently to make sure you don't run your protein off the gel.
Staining, Destaining, & Visualization
- dye overnight or cycles of 1 min @ power 6 in the microwave
- microwave by Bo's bench. Let it vent a little in the hood between heating events.
- Return dye to container
- Rinse to remove residual dye
- Destain (I do 2 rounds at least)
Other Resources
- Background (Wikipedia)
- Instructions for PageRuler protein standard
- Instructions for BioRad Rainbow Std
- Mini-PROTEAN® Tetra Cell manual
Mistakes to Be Careful About
- letting the gel dry too long after pouring the stacking gel (comb step)
- the very edges can shrivel up, which becomes a problem when you try to use those edge lanes
- sample sloshing out of the well you are using into a neighboring well
Recipes
All recipes except the staining & destaining solution are from the Mini-PROTEAN® Tetra Cell manual
Loading Buffer (5x):
- You will mix pre-made 5X loading buffer with fresh beta-mercaptoethanol prior to each use.
- 5x loading buffer:
- mix 50 uL beta-mercaptoethanol to 950 uL sample buffer prior to use.
- Scaled back 10x: 10 uL beta-mercaptoethanol + 190 uL 5x buffer
- Dilute the sample at least 1:2 with sample buffer and heat at 95oC for 4 min to lyse the cells.
- Amanda mixes 160 uL 5X buffer, 8 uL beta-mercapto-ethanol, 32 uL H2O. Use 10 uL per 25 uL of cells.
- ?? Should add up to 8M urea for really hydrophobic proteins
Running Buffer:
- 10x SDS running buffer (1 liter): 30.3 g Tris-HCl, 144 g Glycine, 10 g SDS. Fill to 1 L with ddH2O. Don't add acid or base to adjust pH.
- Make 1L of 1x for use.
- Store at 4oC.
- This buffer is used while running proteins through the gel. Pour it in as the instructions for the box explain. Pour back into bottle for re-use afterward.
- Keep until ____; remake after this.
Staining Buffer(Coomassie Brilliant Blue G-250):
- 0.500 g Brilliant Blue, 500 mL methanol, 100 mL glacial acetic acid, 400 mL dH20. Mix well. Store at room temperature; can be reused 2-3 times.
- note: Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two methyl groups. We use the G form. Read more about the R form here.
Destaining Buffer:
Acrylamide toxicity
- Acrylamide is toxic to your nervous system, and may be a carcinogen. The unpolymerized form is toxic, but the polymerized form is much less toxic. ALWAYS wear gloves and wipe up spills - once the solution drys, the dust can be inhaled. Interestingly, fried starchy/sugary foods naturally contain acrylamide, too.
- more than you want to know about acrylamide toxicity can be found here