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> Western Blotting

> Western Blot Buffers



The best review of protein blotting I have seen is: Gershoni & Palade, Analytical Biochemistry 131: 1-15, 1983. The most commonly used blotting technique is outlined here. The four primary steps are:
1) electrophoretic separation
2) transfer to nitrocellulose (electrophoretic)
3) staining and/or hybridization
4) immunologic identification of proteins on the nitrocellulose

Western Blotting Protocol

1) Separate proteins by electrophoresis in gel. DO NOT DESTAIN GEL.
2) Prepare paper. Immerse in transfer buffer for 5 minutes (see note 3).
i) Ensure transfer tank is full. Transfer buffer may be used for 10 transfers. Mark each use on the outside of the tank.
ii) Load gel and nitrocellulose into holder. Layers from top to bottom: 1 - foam pad 2 - 2 sheets of filter paper (gel drying)* 3 - gel (presoak in transfer buffer 5 min) 4 - nitrocellulose sheet** 5 - 2 sheets of filter paper 6 - foam pad Assemble completely submerged and make sure there are no air bubbles between layers. * S + S #470 ** S + S BA85 (or BA83 for proteins with MW less than 20 kd)
iii) Put holder in the tank and charge the plates. - Remember transfer is towards the anode (+, red) from SDS gels but may be either direction fron non-denaturing gels. Always make sure the nitrocellulose is on the side of the gel that the protein will be transferring toward.
iv) Transfer takes place in 2 hr at 350 mA constant current when using the BioRad minitransblot assembly. Read the directions for this apparatus and then ask for a demonstration before using it.
4) Remove nitrocellulose, stain gel with Coomassie to ensure transfer was even and complete. If you want to protein stain all of or a strip off the edge of the nitrocellulose, do so now.
5) Seal nitrocellulose in bag with blocking buffer. Block for 2-12 h at room temperature with gentle rotation.
6) Wash with Ab buffer 2 x »10 ml.
7) Immunoblot in 10 ml of antibody buffer and 5-10 ml of high titre antiserum 2-6 h at room temperature. Alternatively, dilute the primary antibody to 1-2 mg/ml in 20 mM Tris HCl pH 8.2, 0.1% BSA, 1% normal goat serum. Use 1 ml of this solution for every 10 cm2 of nitrocellulose.
8) Wash the blot 3-5 x with 10 ml wash buffer.
9) To visualize your bands use a labelled second antibody such as second Ab gold or I125 second Ab (in Ab gold hybridization buffer or Ab hybridization buffer). For 125I wash 3 x with wash buffer. Dry and wrap in Saran wrap. When exposing film use an intensifying screen.
10) Silver enhancement. After Ab gold labelling proteins, bands will be pink in colour. Silver enhancement greatly increases sensitivity and makes the bands black. Whether or not you use silver enhancement first wash the blot once with wash buffer. Then wash membrane in distilled water at least 2 x 5 min 20 ml each. Use Boehringer Mannheim silver enhancement kit Intense. Follow the directions. Use dedicated glassware, i.e. glassware used only for silver staining. Rinse all glassware with distilled deionized water before use.
11) Alternatively you can visualize your bands using an alkaline phosphatase conjugated secondary antibody. Add secondary antibody (4 ml) as before with primary antibody. Agitate overnight (» 12 hrs).
12) Wash 3-5 X with 10 mL wash buffer.
13) Wash 1 X with alkaline phosphatase (AP) buffer.
14) Add 10 mL AP buffer Add 66 ml NBT substrate Add 33 ml BCIP substrate Agitate. Development requires 1-15 minutes.
15) Stop the development by rinsing with dH2O.