Bridges Lab Protocols

Preparing a SDS-PAGE Gel: Difference between revisions

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" !...'
 
Reddj (talk | contribs)
164 edits
added SOP requirement-JR
 
(3 intermediate revisions by one other user not shown)
Line 1: Line 1:
==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 || 4 mL || 5 mL
| [[SDS-PAGE Separating Gel Buffer]]|| 3 mL || 3 mL || 3 mL || 3 mL
|-
|-
| [[Acrylamide Solution]] || 3 mL || 3 mL || 3 mL
| [[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) || 200 uL || 200 uL || 200 uL
| APS (make fresh, 0.1g/mL in water) || 200 uL  
|-
|-
| TEMED || 30 uL || 30 uL || 30uL
| TEMED || 30 uL  
|}
|}


[[Category: SDS-PAGE]]
[[Category: SDS-PAGE]]
[[Category: Electrophoresis]]
[[Category: Electrophoresis]]
[[Category: PhosTag]]
[[Category: Phosphorylation]]
Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php/Preparing_a_SDS-PAGE_Gel"