In this step, proteins will be moved from the gel to the membrane. There are three main methods for transferring: tank transfer systems, semi-dry systems, and rapid-transfer systems. In this post, semi-dry systems will be written in detail.
Procedure
Dissemble the gel cassette and remove the stacking gel. Separating gel is needed for transfer.
Immerse thick blot papers, PVDF, and gel in 1X transfer buffer for 10 min. Note1. PVDF must be soaked in 100% methanol for a couple of sec. Make sure membrane was completely wet. Note2. Other membranes can be used instead of PVDF membrane (see PVDF vs Nitrocellulose vs Nylon)
Place the thick blot paper, PVDF, gel, and another thick blot paper (see figure 1). Note. Ensure there are no bubbles between PVDF and the gel. Bubbles disturb transfer. To remove the bubble, once you place the gel on the PVDF, gently remove the bubbles with rollers (Pastuer pipet can be used as rollers). If a roller is too dry, sometimes it tears the gel. Thus, apply a bit of transfer buffer on the roller before using so that you can roll it without tearing the gel.
Transfer at 90 mA for 60 min to transfer proteins size of 10-100 kDa. Note. The voltage and the time can be adjusted.
Figure 1
PVDF vs Nitrocellulose vs Nylon
Most labs use either PVDF or nitrocellulose membranes for western blotting. Nylon membranes can be used in certain circumstances. Nylon membranes can be used for western blotting but it’s more suitable for Southern blotting or northern blotting.
Transfer Buffer for Semi-dry Transfer
Methanol promotes dissociation of SDS from the proteins and improves protein binding capacity onto membranes. Also, prevents the gel from swelling during the transfer.
Transfer Systems
There are three main transfer systems: wet transfer, semi-dry transfer, and dry transfer systems.
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