Chapter 5:
Procedure 5.4

Western Blotting

Background

The gel staining methods described in the previous procedures are methods that stain all proteins. Identification of a specific protein must be verified by more specific techniques, such as immunoblotting, also called Western blotting.

Western Blotting is a method for identifying proteins that combines the resolving power of PAGE with the specificity of antibodies. Because of the power of this technique, Western blotting has become an increasingly important tool, not only for protein chemists but also in clinical labs where Western blotting is now considered the "gold standard" for identifying HIV-positive individuals.

Western blotting is similar to Southern (DNA) and Northern (RNA) blotting in that target molecules are first separated on a gel and then transferred from the gel to a membrane, or blot, which binds the molecules. The membrane is then incubated with reagents that specifically react with target molecules on the membrane. In Western blotting, proteins are detected instead of nucleic acid and the reagent used to detect proteins is an antibody, also a protein, instead of a nucleic acid probe. This antibody specifically binds the target protein. In our case, we will use an antibody to ß-galactosidase, our target molecule of interest. The antibody is visualized because it is labeled by a tag that makes it visible, either by radioactive, chemiluminescent , enzymatic or other means. Most often the “tag” is actually on a second antibody, an antibody that recognizes the first antibody to ß-galactosidase.

Try to make an educated guess as to which bands on your Coumassie-stained gel are ß-galactosidase. The Western blot that you will do now will either prove or disprove your hypothesis regarding the identity of the band(s).

Figure 5.3 The concept of Western blotting. CAUTION: This figure is misleading because the gel is not stained before transferring to a blot.

Separation and Transfer of Proteins

1. Run all your samples on a PAGE gel just as you did in the previous procedure, beginning with the crude extract and all the succeeding purified fractions. Load the same amounts of protein that are appropriate for Coomassie Blue detection. Also run at least one ß-galactosidase standard purchased from a commercial supplier and a lane of molecular weight standards. It is preferable to use pre-stained molecular weight standards as they make it easy to determine that the transfer has occurred.
2. The membrane used for the blot is nitrocellulose. Cut one sheet of nitrocellulose and four sheets of Whatman 3MM filter paper to the size of the gel. Always handle membranes with gloves.
3. Soak the nitrocellulose membrane in distilled water. Nitrocellulose should be wetted by carefully laying it on the surface of the water. Allow the nitrocellulose to wet by capillary action for several minutes and then submerge the sheet for 2 minutes. Move the membrane to transfer buffer for 5 minutes. Wet the filter paper by soaking it in transfer buffer. The recipe for this buffer and the other buffers used in this procedure are given in appendix A.
4. Immerse the gel, membrane, filter papers and support pads in transfer buffer to be sure they are thoroughly soaked.
5. Assemble the transfer sandwich so that the membrane is in direct contact with the gel, making sure there are no air bubbles trapped between the gel and the nitrocellulose. Keep all the components wet and make sure the sandwich is tightly assembled. The assembly is shown in Figure 5.4

Figure 5.4 Arrangement of the gel, blot, filter paper and support pads for electrophoretic transfer of proteins for a Western Blot.




6. Place the complete sandwich in the transfer tank with the membrane closest to the anode or positive (red) electrode. Fill up the transfer tank with buffer, making sure that the gel and blot are completely submerged. Put a stir bar in the bottom of the tank.
7. The rate of transfer of proteins is a function of their molecular weight. Larger proteins generally require higher voltages and longer transfer times than smaller proteins. A convenient and reliable method is to transfer overnight. We will follow the recommendations that accompany the Trans-Blot unit, 30 volts at 40 mA, for overnight transfer. [If you want to do your transfer in a shorter time, consult the Trans-Blot (or your equipment) literature.]
8. Disconnect the power supply. Disassemble the sandwich and mark the membrane by clipping off approximately 1 cm of the upper left hand corner. Place the blot into PBS and rinse several times.

Blocking the Membrane

9. Place your membrane in blocking buffer (3% BSA or nonfat dried milk in PBS) and incubate at room temperature for 2 hours with agitation or leave the blot overnight at 4°C in blocking buffer. The purpose of blocking is to keep the membrane from adsorbing antibody molecules nonspecifically. The membrane has a high affinity for protein and we want the antibody to bind only at the sites where the ß-galactosidase is located. What would happen if we forgot to block the membrane or, if for some reason, the blocking step did not work?

Antibody Binding

10. Rinse the membrane twice for 5 minutes each in PBS.
11. Prepare the first antibody, the antibody against ß-galactosidase by diluting it according to the supplier directions in a solution of 3% BSA/PBS (usually l/2000 dilution or greater). The volume of the antibody solution should be enough to cover the blot. Large plastic weigh boats (about 4 inch square) work well and require about 20 mls. **Note: To minimize the volume of solution needed, the blots can be sealed in plastic wrap with heat sealable plastic bags (Fisher). Immerse the membrane in the diluted antibody solution. The anti-ß-galactosidase antibody is a mouse monoclonal antibody that was prepared by a commercial supplier.
12. Incubate at room temperature for 1 hour with agitation. (Can be overnight if necessary)
13. To remove unbound antibody, wash the blot with four changes of Tris buffered saline (TBS) for 5-10 minutes each. Note that we are also changing the buffer in this step. TBS is the preferred buffer for the steps that follow.
14. Prepare the secondary antibody solution. The secondary antibody is a conjugate that consists of an antibody that recognizes and binds to antigenic determinants on the mouse anti-ß-galactosidase and has the enzyme, alkaline phosphatase, coupled to the antibody. The enzyme and the antibody are each prepared and purified separately from different sources by the supplier. They were then conjugated chemically by covalently coupling the enzyme to the antibody. Dilute the conjugate as directed by the supplier in a solution of 3% BSA/TBS (usually at least 1/1000 dilution.)
15. Immerse the membrane in the diluted solution of conjugate. Incubate 1 hour at room temperature with agitation.

Detection

16. To remove unbound conjugate, wash the blot with four changes of PBS for 5-10 minutes each.
17. Prepare the alkaline phosphatase substrate solution and use 10 ml per blot. The substrate solution contains the substrate for alkaline phosphatase, bromo-chloro-indolyl-phosphate (BCIP) and a dye, Nitro Blue Tetrazolium (NBT). This solution is light sensitive and should be made the day of use and stored at 4°C. Develop blots at room temperature with agitation until the bands are suitably dark. Cover the blots with foil while they are developing. Check often as the reaction can happen quickly but can take 30 minutes. Expect to see a brownish-purple-colored band in lanes where ß-galactosidase has been loaded.
18. To stop the reaction, rinse with a PBS solution containing 20 mM EDTA.
19. To save the blot dry it on a sandwich of filter paper, between paper towels, away from the light.

Contact Us:

Lisa Seidman
lseidman@matcmadison.edu
(608) 246-6204

Jeanette Mowery
jmowery@matcmadison.edu
(608) 243-4307