- Data Analysis
- Other Protocols
Western Blot Transfer
- Remove gel from the gel-cast and place into Transfer buffer. (Remember, if you are using bis-tris gels, the gel needs to be equilibrated in the transfer buffer for 10 minutes. If you don’t do this, the transfer will be less efficient because the MOPS-SDS buffer is different than the tris-glycine-SDS-methanol buffer used for transfer.)
- Cut filter paper in half and soak in transfer buffer (You need six halves per gel.) Most commercially available filter papers will work. The piece of filter paper that you use needs to be larger than your gel on all sides.
- Cut a piece of PVDF membrane for transfer and soak in methanol for at least one minute.
- Place three pieces transfer-buffer soaked filter paper on the transfer apparatus (roll out any air bubbles every time).
- Take the membrane from out of the methanol and rinse it in transfer buffer. Although there is 20% methanol in the transfer buffer, this step washes away excess methanol so that proteins will bind the membrane more efficiently. The membrane should be rinsed in transfer buffer until the buffer no longer beads off the membrane.
- Place the membrane onto the three pieces of filter paper and gently roll out any air bubbles.
- Place the gel on top of the membrane in the correct orientation. The marker should be on the same side that it was when you loaded the gel.
- Gently roll out any air bubbles.
- Place three more pieces of filter paper on top of the gel. Gently roll out all air bubbles.
- Set the transfer apparatus to 15V for 45 minutes and hit start.