In general, we recommend including 20% methanol in transfer buffer, and performing a wet transfer for 2 hr at 70 V. (Do not do this if using Trans-Blot® Turbo system) Power conditions were too high or transfer time too long (proteins may transfer through the membrane and into the filter paper) Shorten transfer time; Reduce transfer voltage; Transfer buffer was incorrect or prepared incorrectly There are two common membrane types used for western blot analysis: PVDF and nitrocellulose. It is never a bad idea to look for a few bubbles (a sure sign that SOMETHING is happening). Do not just hit “transfer” and walk away. Long transfer times at a constant voltage lead to unwanted heat, decreasing resistance, and increasing current. There are two times during a Western blot when an electric current is applied: during the initial “running” step (SDS-PAGE) and during the transfer step (sometimes called the “blotting… 4. Transfer the gel (save the dye mixture; it can be re-used many times) to a mixture of 67.5% distilled water, 7.5% acetic acid, and 25% methanol, place on shaker, and replace with fresh rinse mixture until the excess dye has been removed. PROTEIN TRANSFER. If you are new to Western blotting — or trying a new protocol for the first time — you’ll need to optimize the electrophoresis conditions.. Incubate for 4 h to overnight at room temperature on a shaker. Stay a few minutes to make sure that your voltage and amps are okay. Incomplete transfer can be corrected by longer transfer or by the use of higher voltage. Disappearing Transfer. Equilibrate gel in transfer buffer for 10 minutes prior to transfer. An overview of western blotting transfer methods, including blotting membranes ... (5 to 30 V) from as little as 1 hour to overnight. Transfer speed ranges from 1 h to overnight, usually at a constant voltage to maintain field strength and maximize efficiency of transfer. Transfer is performed by passing a current across the gel to the membrane. Comparison of transfer methods in Western blotting. After electrophoresis is complete, proteins must be transferred from the gel onto a suitable membrane for antibody staining and detection. Transfer and Membrane. Watch the voltage and amps.