User:Torsten Waldminghaus/protein gel western: Difference between revisions

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(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 |...)
 
 
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*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
|}

Latest revision as of 03:39, 12 November 2010

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
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