Western Blotting
(semi-dry blot)
- store the blotting apparatus with a paper towel placed between the electrodes
- before use place a wet paper towel between the electrodes for 10 min.
- cut per blot 6 pieces of Whatman paper and one piece of Nitrocellulose (i.e. Osmonics Inc., Nitrobind, 0.45 µm), all gel-size
- incubate Nitrocellulose for at least 1 min in water
- set up blot:
Anode - three wet Whatman - gel - Nitrocellulose - three wet Whatman - Kathode
(the Whatman paper gets wetted in Transfer-buffer)
(remove airbubbels by rolling over the setup with a 25 ml glaspipett) - blot for at least 45 min. with 0.7 mA/cm2 (i.e. 50 mA for one Biorad Miniprotean gel)
- stain the blot with PonceauS solution, 10 sec. to 1 min.
- destain with water
- mark marker bands, slots, orientation
- block in blocking solution for 45 min. (room temperature) to overnight (coldroom)
- incubate with 1. antibody for at least 45 min. (room temperature)
- wash 4 times with 1 x Rinse-buffer, 5 min. each
- incubate with 2. antibody for at least 30 min. (room temperature)
- wash 4 times with 1 x Rinse-buffer, 5 min. each
- incubate with ECL developing solutions (i.e. NEN Renaissance, Roche Lumi-Light)
- place on glas plate, cover with Saran
Wrap, expose to film
- stripping
and reprobing of blot:
incubate 2 x 15 min. in stripping-buffer, 2 x 15 min. in 1 x Rinse-buffer and proceed with blocking step
Buffers:
Transfer-buffer:
48 mM Tris; 39 mM glycine; 0.4 % SDS; 20 % Methanol.
[for 1 l: 5.8 g Tris; 2.9 g glycine; 4 g SDS; 200 ml Methanol]
Ponceau S solution:
0.1 % in 1 % acetic acid (v/v) (can be reused many times)
Blocking solution:
1 % BSA; 1 % Ovalbumin; in 1 x Rinse-buffer; sterile filter.
10 x Rinse-buffer:
[for 500 ml: 87.66 g NaCl; 20 ml 2 M Tris/HCl; 5 ml 2M
Tris/base; 25 ml Tween20; sterile filter]
10 x Stripping-buffer:
1 M glycine pH 2.5/HCl
