User:Torsten Waldminghaus/protein gel western

From OpenWetWare

< User:Torsten Waldminghaus(Difference between revisions)
Jump to: navigation, 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 |...)
Current revision (05:39, 12 November 2010) (view source)
(Recipe for buffers)
 
Line 29: Line 29:
|}
|}
*Store at -20°C
*Store at -20°C
-
*Should be 15% of endvolume (for example 42.5 μL sample + 7.5 μL Loading buffer
+
*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
 +
|}

Current revision

Recipe for buffers

Resuspension buffer
20 ml TE buffer
5 ml Stacking gel buffer
3.5 ml 10% SDS
1.5 ml glycerol
4 ml ddH2O
  • Store at rt.


Loading buffer
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

  1. 2 filter papers with Anode buffer I
  2. 1 filter papers with Anode buffer II
  3. Membrane
  4. Gel
  5. 3 filter papers with Cathode buffer
  • Run 30 min at 15V


Anode buffer I
36.3 g Tris
200 ml methanol
H2O to 1 liter


Anode buffer II
3.03 g Tris
200 ml methanol
H2O to 1 liter


Cathode buffer
5.2 g 6-amino-n-hexanoic acid
200 ml methanol
H2O to 1 liter
Personal tools