In semi-dry Western Blot, the electrodes are placed directly in contact with the gel/nitrocellulose membrane sandwich to provide a fast, efficient transfer. The polyacrylamide gels must be equilibrated in transfer buffer, to remove electrophoresis buffer salts and detergents, and the nitrocellulose membranes and filter papers arepre-wetted, but that is all the buffer that is required. It is important to exclude excess moisture and air bubbles trapped in the filter papers and membrane when setting up the transfer, usually a pipet rolled over the surface will take care of this, but other than that, the set-up for this process is extremely simple.
• Make 1X transfer buffer freshly.
A recipe for 1X transfer buffer (48 mM Tris base, 39 mM glycine, 20 % methanol):
|For 1.0 L:||5.76 g Tris-base
2.95 g glycine
200 mL methanol
|Add ddH2O to 1 L.|
• PVDF or nitrocellulose membrane.
• Gel sized filter papers.
• Standard liquid based polyacrylamide gel transfer system.
|1||Prepare in advance the nitrocellulose and filter/blot paper.|
|2||After running an SDS/PAGE gel, immediately equilibrate the gel in a small container of Semi-dry transfer buffer for ~15min.|
|3||Completely saturate a piece of blot paper by soaking in transfer buffer. Place this pre-soaked sheet of blot paper onto the platinum anode. Roll a pipet or test tube over the surface of the paper (like a rolling pin) to exclude all air bubbles.|
|4||Carefully place the equilibrated gel on top of the nitrocellulose membrane, aligning the stack as perfect as possible.|
|5||Soak another piece of blot paper and place on top of the gel, carefully removing air bubbles from between the gel and filter paper.|
|6||Carefully place the cathode plate onto the stack. Press to engage the latches with the guide posts without disturbing the gel/nitrocellulose stack.|
|7||Run the transfer unit at 320mA for 1 hour.|
|8||When the transfer is complete, turn off the power and peel off the layers of the sandwich until you reach the membrane. Remove the membrane with forceps and rinse it in deionized water.|