A Western-blot showing b-actin from rat hepatocytes resolved using 10% in-house SDS-PAGE and Lonza ProSieve EX running buffer and transferred using 1X ProSieve EX Western transfer buffer.
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This product from Lonza is a modified Towbin Western blot transfer buffer that decreases the transfer time to around 20 minutes without compromising protein transfer on to nitrocellulose membrane.
Western blot transfer of proteins from protein gels to nitrocellulose membranes.
Rat primary hepatocyte lysates resolved on SDS-PAGE gels
After resolving the protein on SDS-PAGE, the gels were soaked for 5 minutes in distilled water followed by soaking in the 1X ProSieve EX Western transfer buffer for 5 minutes. Nitrocellulose membranes and pads were also soaked in 1X ProSieve EX Western transfer buffer for 5 minutes . The transfer assembly was prepared as usual for tank blot using the Bio-Rad Mini Trans-Blot module. The tank was then filled with 1X 1X ProSieve EX Western transfer buffer with the chiller-block in place and transfer initiated by setting the current at 375 mA for 20 minutes.
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Good transfer is achieved in 20 minutes using the ProSieve EX Western transfer buffer as the markers (perstained) were completely transferred onto the blot without any residual markers left on the gel. We routinely use the membrane to probe at least 3 proteins (e.g. Akt, Gsk3 and b-Actin). We are able to strip and reprobe at least twice with good signal the 3rd time too, indicating the transfer from the gel to the membrane was efficient.
The 1x buffer can be reused. In our hands, the buffer worked well when used up to 6 times without loss of transfer efficiency. Used 1x buffers were stored at room temperature. After repeated use, the buffer may turn a slight brown color, but no loss in transfer efficiency was seen. When using the buffer 3 to 6 times, transfer time increased from 20 to 35 minutes; the desired level of protein transfer to the nitrocellulose membrane was still achieved. When used in combination with ProSieve EX running buffer, samples can be resolved on SDS-PAGE and transferred onto nitrocellulose membrane within 1 hr and 15 min.