Western blot transfer can be done in wet or semi-dry conditions. In wet western blot transfer (tank transfer), the wet western blot transfer mode is a sandwich with a regular order. That's a sandwich of sponge/paper/gel/membrane/paper/sponge, which is clamped tightly, no air bubbles within it. It's important that the gel is closest to the negative electrode and membrane closest to the positive electrode. Semi-dry western blot transfer is generally faster but wet western blot transfer has a less tendency to failure and is especially recommended for large proteins more than 100 kD.
|For 1.0 L:||3.0 g Tris-base
14.4 g glycine
200 mL methanol
|Add ddH2O to 1 L.|
NOTE: for the proper transfer of large proteins, up to 0.5% SDS may need to be added to 1X transfer buffer.
|For 1.0 L:||30 g Tris-base
144 g glycine
|Add ddH2O to 1 L.|
NOTE: To make 1X transfer buffer from 10X: Mix 100 ml of 10X transfer buffer, 200 ml of methanol and 700 ml of ddH2O per liter.
|1||Run the samples in SDS-PAGE as usual.|
|2||Prepare and mark the membrane (right size, PVDF or nitrocellulose membrane) with a pencil; then soak it in Methanol for a few minutes, and rinse it with distilled water.|
|3||Equilibrate the membrane, pads, filter papers (4 pieces) and transfer foam in Transfer Buffer (store in 4°C) for 5 minutes.|
|4||Assemble transfer sandwich in the following order: black frame (negative electrode) >> foam >> 3 pieces of filter papers >> SDS-PAGE gel >> membrane >> 3 pieces of filter papers >> foam >> red frame (positive electrode). Make the marked side of membrane face the gel. Put it into transfer tank.|
Fig 2. Schematic picture of Western Blot wet transfer sandwich
|5||Transfer the gel at 80 V for 90 mins in cold room. Alternatively, use ice bag and magnet stirring bar to transfer the gel in room temperature.|
|6||Disassemble the gel pack, and use a pencil to mark the well imprints on the membrane.|
For wet western blot transfer, generally, the current is 1-2 mA/cm2 depending on the membrane size, but 200 mA is usually applicable in most laboratories. Long transfer time is more suitable for tank systems, which normally require cooling of the unit and internal recirculation of the transfer buffer; in semi-dry transfer, however, prolonged blotting may result in buffer depletion, overheating and gel drying. The table below is for your information about how to decide the wet transfer time, but note that you can make proper changes according to actual situation.
|Molecular weight of target protein (kDa)||Transfer time (h)|
|80 - 140||1.5 - 2.0|
|25 - 80||1.5|
|15 - 40||0.75|