实验概要

Peprotech provides a typical western transfer and development protocol.

实验原理

Western Transfer, also known as Western Blotting, is a rapid immunoblotting technique for identifying the presence of a particular protein in a complex mixture of proteins such as cell lysates or sera. The technique exploits both the efficiency of SDS-PAGE to separate a mixture of proteins into distinct protein bands, and the ability of immunochemical reagents to interact specifically with a given protein antigen.

主要试剂

1. A nitrocellulose membrane, approximately the size of the gel, presoaked in Western Transfer Buffer* for five minutes. (Note: The membrane should be handled with gloves and clean forceps to avoid contamination with extraneous proteins.)

2. *Western Transfer Buffer (1/2 x TOWBIN)
Tris Base 1.45g (12mM final concentration)
Glycine 7.2g (96mM final concentration)
Methanol 200mL (20% final volume)
diH₂O add to 1.0L final volume

3. Antigen specific probing antibody (PrimaryAntibody)

4. Secondary antibody (Donkey-anti-probing antibody species conjugated to Alkaline Phosphatase)

5. Commercially available alkaline phosphatase conjugate substrate kit

主要设备

1. SDS-PAGE apparatus and accessories

2. Submerged Western Transfer Cassette and accessories

实验步骤

1. Load the protein sample onto a 4-20% Tris-Glycine polyacrylamide gel and run until desired resolution is achieved. (The electrophoresis can be followed by using pre-stained molecular weight markers).

2. Set up the Submerged Western Transfer Cassette as described below and in Figure III-2:

    1) Submerge the open Transfer Cassette cathode plate onto the tray pre-filled with Western Transfer Buffer.

    2) Place a sponge support pad onto the cassette and remove the air bubbles by gently rolling a Pasteur pipette over the pad.

    3) Place a piece of blotting paper onto the sponge support pad.

    4) Remove the gel from the electrophoresis plates, cut off approximately the bottom 3mm of the gel so that the membrane can be laid flat against the gel, and place the gel over the blotting paper. Expel air bubbles as before.

    5) Carefully place the pre-soaked nitrocellulose membrane onto the gel and expel air bubbles. Ensure that the membrane remains directly over the gel before proceeding.

    6) Place a second piece of blotting paper onto the nitrocellulose membrane and remove air bubbles.

    7) Place a sponge support pad onto the second piece of blotting paper and remove air bubbles.

    8) Gently close the cassette by placing the anode plate over the exposed pad.

3. Carefully place the assembled cassette into the transfer tank containing Western Transfer Buffer up to the "pre-fill" level and adjust the buffer level, as needed, after the addition of the cassette.

4. Connect the assembled apparatus to an electrophoresis power supply and run for approximately 1.5 hours at a constant current of 400mA.