After proteins have been separated by electrophoresis, individual protein bands can often be identified by using an antibody that is specific to that protein. However, to be accessible to antibodies the proteins in the gel must first be transferred from the gel to a membrane. This process is called "Western blotting" (after E. M. Southern). We will use the "semi-dry" method of Western blotting to transfer proteins from our gels to a nitrocellulose membrane. The semi-dry method can be used to rapidly transfer up to six gels at a time, and it does not require a large power source. In the original description, different buffers were used for the anode and cathode, but recently it has been demonstrated that it is just as effective to use a slightly diluted version of the Tris/glycine running buffer used for SDS-polyacrylamide gels.
Materials (for one transfer)
|Transfer Buffer||Concentration||for 1000 mL|
|Methanol||20% (v/v)||200 mL|
|Tris base||25 mM||3.63 g|
|Glycine||192 mM||14.4 g|
|Distilled water||--||up to 1000 mL|
Multiple gels can be transferred on the same apparatus by stacking
up to six of the complete "modules" of blotting paper/nitrocellulose membrane/gel/blotting
paper. A dialysis membrane (presoaked in transfer buffer) is used
to separate each module from the one below. A dialysis membrane is
not added to the top of the final module because direct contact between the
electrode and the absorbent paper is required.
The type of bonds that hold proteins to nitrocellulose are not known; however the binding is blocked by oils or other proteins. Wear gloves at all times when handling the nitrocellulose!
1. Rinse the electrode plates of the semi-dry apparatus with distilled H2O.The polyacrylamide gel can now be stained to verify transfer. Pre-stained molecular weight standards run on the gels should transfer to the nitrocellulose, and will serve as internal markers for transfer as well as for molecular weight estimation.
2. Collect four sheets of absorbent paper (Whatman 3MM or equivalent) and one sheet of nitrocellulose.
3. Soak the nitrocellulose membrane in distilled water. Nitrocellulose should be wetted by carefully laying it on the surface of the water, and allowing the nitrocellulose to wet from the bottom by capillary action.
4. After wetting, submerge the membrane for 2 minutes.
5. Wet the absorbent paper by soaking in the transfer buffer shown below.
6. Open the polyacrylamide gel container and trim off the stacking gel.
7. Orient the gel by making a mark or cut in the lower left-hand corner, below the number one lane.
8. Briefly rinse the gel with H2O.
9. Assemble each module, in the following order, starting from the bottom electrode plate (the anode);
10. As you assemble the stack, check carefully for air bubbles and gently remove them, either by using a gloved hand or by rolling a glass rod over the sandwich.
- two layers of thick absorbent paper, thoroughly soaked in transfer buffer
- a wet nitrocellulose membrane
- the polyacrylamide gel
- two layers of thick absorbent paper, soaked in transfer buffer
11. After adding the gel, remove any excess buffer from around the gel and surround it if necessary with plastic strips, 1 - 2 inches wide, to prevent the current from short-circuiting around the gel.
12. Above the top two layers of absorbent paper lay a sheet of dialysis membrane before starting the next module.
13. After the last module has been assembled carefully place the upper electrode (the cathode) on top of the stack, omitting the dialysis membrane.
14. Connect the electrodes (positive or red lead to the bottom) and begin the transfer. Running times are 45 min to 1.5 hr with a current of 0.8 - 1.0 mA/cm2 of gel. Avoid extending the running time, as this can cause the gel to dry, or proteins to transfer through the nitrocellulose.
15. After transfer, disconnect the power source.
16. Carefully disassemble the stack, placing the membrane on dry Kimwipes and store at room temperature*.
*Several workers recommend that the membrane be allowed to dry
completely after blotting, before further processing the nitrocellulose.
In some cases, this seems to lessen the chance of the transferred proteins
being lost in subsequent steps. An alternative to drying is to treat
the membrane with isopropanol for one minute.