Why should we carry the Western blot in the low temperature while we were transferring SDS-PAGE information to the PVDF membrane?
Carrying out the Western blot at a low temperature is important because it helps prevent the denaturation of proteins during the transfer of SDS-PAGE information to the PVDF membrane. The low temperature minimizes the movement and unfolding of the proteins, ensuring that they retain their native structure on the membrane. This is critical because the Western blot relies on the detection of specific proteins using antibodies that recognize their native conformation.
To perform the Western blot at a low temperature during the transfer step, you can follow these steps:
1. Prepare an ice bath or use a cold room set to around 4°C. Keeping the temperature low will help maintain the stability of the proteins.
2. After completing the SDS-PAGE gel electrophoresis, carefully remove the gel from the cassette and rinse it briefly in transfer buffer. The transfer buffer typically contains Tris and glycine, as well as a small percentage of methanol. It is recommended to pre-chill the transfer buffer to the desired low temperature as well.
3. Wet the PVDF membrane and the stack of blotting papers in transfer buffer. Ensure that the membrane is thoroughly soaked but not oversaturated.
4. Assemble the transfer sandwich in the following order: one piece of wet blotting paper, the PVDF membrane, the gel, and another piece of wet blotting paper on top. Make sure that there are no air bubbles trapped between the layers.
5. Place the transfer sandwich into a transfer apparatus (typically a semi-dry or wet transfer system) that is cooled by the ice bath or kept in a cold room. Ensure that the electrodes and the transfer apparatus are in good contact and secure the sandwich in place.
6. Begin the transfer process at a constant low voltage or current, as per the manufacturer's recommendations or the specific experimental requirements. The transfer time will depend on the size and type of proteins being transferred.
By carefully following these steps and conducting the Western blot at a low temperature, the risk of protein denaturation and loss of antigenicity during the transfer process will be significantly reduced, resulting in more accurate and reliable results.