User:Torsten Waldminghaus/protein gel western: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: ==Recipe for buffers== {|border="1" |+ '''Resuspension buffer''' |- |20 ml TE buffer |- |5 ml Stacking gel buffer |- |3.5 ml 10% SDS |- |1.5 ml glycerol |- |4 ml ddH<sub>2</sub>O |...) |
|||
Line 29: | Line 29: | ||
|} | |} | ||
*Store at -20°C | *Store at -20°C | ||
*Should be 15% of endvolume (for example | *Should be 15% of endvolume (for example 30 μL sample + 5.3 μL Loading buffer | ||
==Western== | |||
*activate the membrane (for example Immobilin -P, Millipore) with methanole and then equilibrate with water. | |||
===Semi dry blot assembly=== | |||
#2 filter papers with Anode buffer I | |||
#1 filter papers with Anode buffer II | |||
#Membrane | |||
#Gel | |||
#3 filter papers with Cathode buffer | |||
*Run 30 min at 15V | |||
{|border="1" | |||
|+ '''Anode buffer I''' | |||
|- | |||
|36.3 g Tris | |||
|- | |||
|200 ml methanol | |||
|- | |||
|H<sub>2</sub>O to 1 liter | |||
|} | |||
{|border="1" | |||
|+ '''Anode buffer II''' | |||
|- | |||
|3.03 g Tris | |||
|- | |||
|200 ml methanol | |||
|- | |||
|H<sub>2</sub>O to 1 liter | |||
|} | |||
{|border="1" | |||
|+ '''Cathode buffer''' | |||
|- | |||
|5.2 g 6-amino-n-hexanoic acid | |||
|- | |||
|200 ml methanol | |||
|- | |||
|H<sub>2</sub>O to 1 liter | |||
|} |
Latest revision as of 03:39, 12 November 2010
Recipe for buffers
20 ml TE buffer |
5 ml Stacking gel buffer |
3.5 ml 10% SDS |
1.5 ml glycerol |
4 ml ddH2O |
- Store at rt.
285 μL 2.5M DTT |
205 μL 10%SDS |
10 μL 10% Bromphenolblue |
- Store at -20°C
- Should be 15% of endvolume (for example 30 μL sample + 5.3 μL Loading buffer
Western
- activate the membrane (for example Immobilin -P, Millipore) with methanole and then equilibrate with water.
Semi dry blot assembly
- 2 filter papers with Anode buffer I
- 1 filter papers with Anode buffer II
- Membrane
- Gel
- 3 filter papers with Cathode buffer
- Run 30 min at 15V
36.3 g Tris |
200 ml methanol |
H2O to 1 liter |
3.03 g Tris |
200 ml methanol |
H2O to 1 liter |
5.2 g 6-amino-n-hexanoic acid |
200 ml methanol |
H2O to 1 liter |