Western blotting

Avoid touching the membranes, use gloves and blunt-ended forceps!!!

The membranes must not be allowed to dry out during any step!!!

Before transfer: precool Transfer buffer!!! Do not forget to activate the PVDF membrane by soaking in methanol for > 1 min, then rinse in water, and soak in Transfer buffer!!! Equilibrate the gel and soak the filter paper, and fiber pads in the buffer. If it is a preparation for the N-terminal sequencing - use only the PVDF membranes designed for protein sequencing, e.g. Sequi-Blot PVDF membrane from Bio-Rad.

Transfer conditions: transfer at > 100 V for > 60 min (< 400 mA).

After transfer: Stain the nitrocellulose membrane with Ponceau S to verify the transfer and to locate e proteins.

1.Block the membrane in TBS-Tween with 2-5 BSA or defatted milk powder for 2 hr (at room temperature) or overnight (at +4C) on a shaker.

2.Incubate with I-Antibody for 2-3 hr at room temperatute or at +37C.
This step can be combined with blocking. Dilute antibody in TBS-Tween with BSA or defatted milk powder. Find optimal dilution for the antibody.

3.Wash 3  10 min with TBS-Tween (> 4 ml/cm²).

4.Incubate with II-Antibody for 1-2 hr at room temperature or at least for 45 min at +37C on a shaker. Dilute antibody in TBS-Tween with BSA or defatted milk powder. The antibodies with coupled peroxidase normally are very sensitive and the dilution 1:20 000 shoul be sufficient. The dilution determines the most part of the background.

5.Wash 3  10 min with TBS-Triton, and leave it in TBS.
The time should be exact!!! Otherwise, Triton washes away everything.

6.Develop the membrane.

Chemiluminescence (peroxidase coupled II-Antibodies) is the most sensitive method. The signal is detected with x-ray film. The reagents: ECL+Plus (Amersham) or home-made ECL solutions. With this method it is possible to strip the plot, and to decorate with up to 4 different antibodies.

Chromogenic method (alkaline phosphatase coupled II-Antibodies): the plot is directly stained and, therefore, the stripping is complicated. The kits are available at Roche.

7. Store the membrane.

For a couple of weeks keep the membrane in TBS. For longer period: in TBS with 0.1 of Na₂NO₃ at +4C.

For all incubations and wash steps the PBS-Tween buffer could be used instead of TBS-Tween.

2004 02 12 – Oliver & Julius