Difference between revisions of "Preparing a SDS-PAGE Gel"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (Created page with '==Separating Gel== *For 12 mL final volume (2 gels) *Combine, adding APS and TEMED last *Pour into cassette, overlay with the top layer of water saturated butanol {| border="1" !...') |
(added SOP requirement-JR) |
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| + | ==SOP== | ||
| + | *[[SOP- Electrophoresis]] | ||
| + | |||
==Separating Gel== | ==Separating Gel== | ||
*For 12 mL final volume (2 gels) | *For 12 mL final volume (2 gels) | ||
| Line 4: | Line 7: | ||
*Pour into cassette, overlay with the top layer of water saturated butanol | *Pour into cassette, overlay with the top layer of water saturated butanol | ||
{| border="1" | {| border="1" | ||
| − | ! Component || 7.5% || 10% || 12.5% | + | ! Component || 6% ||7.5% || 10% || 12.5% |
|- | |- | ||
| − | | [[SDS-PAGE Separating Gel Buffer]] || 3 mL || | + | | [[SDS-PAGE Separating Gel Buffer]]|| 3 mL || 3 mL || 3 mL || 3 mL |
|- | |- | ||
| − | | [[Acrylamide Solution]] || | + | | [[Acrylamide Solution]]|| 2.4 mL || 3 mL || 4 mL || 5 mL |
|- | |- | ||
| − | | Water || 6 mL || 5 mL || 4mL | + | | Water || 6.6 mL|| 6 mL || 5 mL || 4mL |
|- | |- | ||
| − | | APS (make fresh, 0.1g/mL in water) || 200 uL || 200 uL || 200 uL | + | | APS (make fresh, 0.1g/mL in water) || 200uL || 200 uL || 200 uL || 200 uL |
|- | |- | ||
| − | | TEMED || 30 uL || 30 uL || 30uL | + | | TEMED || 30 uL || 30 uL || 30 uL || 30uL |
|} | |} | ||
| + | *For a [[Phos-Tag Electrophoresis and Western Blotting|Phos-Tag Gel]], add 24 uL 5 mM Phos-Tag and 24 uL 10 mM MnCl in the separating gel only | ||
==Stacking Gel== | ==Stacking Gel== | ||
| Line 31: | Line 35: | ||
| Water || 4 mL | | Water || 4 mL | ||
|- | |- | ||
| − | | APS (make fresh, 0.1g/mL in water) | + | | APS (make fresh, 0.1g/mL in water) || 200 uL |
|- | |- | ||
| − | | TEMED || 30 uL | + | | TEMED || 30 uL |
|} | |} | ||
[[Category: SDS-PAGE]] | [[Category: SDS-PAGE]] | ||
[[Category: Electrophoresis]] | [[Category: Electrophoresis]] | ||
| + | [[Category: PhosTag]] | ||
| + | [[Category: Phosphorylation]] | ||
Latest revision as of 16:38, 4 June 2020
SOP
Separating Gel
- For 12 mL final volume (2 gels)
- Combine, adding APS and TEMED last
- Pour into cassette, overlay with the top layer of water saturated butanol
| Component | 6% | 7.5% | 10% | 12.5% |
|---|---|---|---|---|
| SDS-PAGE Separating Gel Buffer | 3 mL | 3 mL | 3 mL | 3 mL |
| Acrylamide Solution | 2.4 mL | 3 mL | 4 mL | 5 mL |
| Water | 6.6 mL | 6 mL | 5 mL | 4mL |
| APS (make fresh, 0.1g/mL in water) | 200uL | 200 uL | 200 uL | 200 uL |
| TEMED | 30 uL | 30 uL | 30 uL | 30uL |
- For a Phos-Tag Gel, add 24 uL 5 mM Phos-Tag and 24 uL 10 mM MnCl in the separating gel only
Stacking Gel
- Rinse polymerized separating gel with water
- For 10 mL (2 gels)
- Combine, adding APS and TEMED last
- Pour over separating gel and insert comb
| Component | |
|---|---|
| SDS-PAGE Stacking Gel Buffer | 2.5 mL |
| Acrylamide Solution | 1.7 mL |
| Water | 4 mL |
| APS (make fresh, 0.1g/mL in water) | 200 uL |
| TEMED | 30 uL |
