Just as proteins have been bound with electrical charge provided by SDS, they can be transferred in an electrical field from the gel onto a sturdy support, a membrane that "blots" the proteins from the gel. This is called western blot transfer. Here we will talk about methods of western blot transfer, transfer membranes, common transfer buffers, and transfer time, etc.
Wet and semi-dry transfer are the two most common electrotransfer methods and provide greater speed and efficiency than alternatives based on diffusion, capillary action, and vacuum. Western blot transfer can be done in wet or semi-dry conditions. Semi-dry transfer is generally faster but wet transfer has a less tendency to failure and is especially recommended for large proteins more than 100 kD. In wet transfer, from the image above, you can see the western blot transfer modle-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.
There are two basic transfer membranes available, nitrocellulose and PVDF (polyvinylidene difluoride). In western blot, either 0.2 µm or 0.45 µm pore size membrane can be chosen according to the size of your target protein molecules. For most applications, PVDF and nitrocellulose are equivalent. Note that PVDF has a higher capacity than nitrocellulose but needs more careful pretreatment: cut the membrane to the appropriate size according to the size of your gel, usually a little larger than the gel; soak it in methanol for about 30 seconds; wash it in ddH2O for about 1 minute, and then incubate in ice cold transfer buffer for 5 minutes. The gel also needs to equilibrate in ice cold transfer buffer for 3 to 5 minutes.
Two common transfer buffer recipes in Western Blotting are:
|1X transfer buffer (wet)||For 1.0 L:||3.0 g Tris-base
14.4 g glycine
200 mL methanol
|1X transfer buffer (semi-dry)||For 1.0 L:||5.76 g Tris-base
2.95 g glycine
200 mL methanol
|Add ddH2O to 1 L.||Add ddH2O to 1 L.|
What are the role of methanol and SDS in Western Blot transfer buffers?
Methanol in transfer buffer helps prevent gel swelling and improves the efficiency of protein binding to membranes (especially nitrocellulose). However, it can also cause reduction in gel pore size, protein charge changes, and protein precipitation. Therefore, it is recommended that one should adjust methanol concentration according to his experiment conditions. Some labs have found that the addition of methanol doesn't increase transfer efficiency significantly, so they replace 20% methanol with 10% ethanol.
SDS (Sodium Dodecyl Sulfate) should not be added into transfer buffers in routine Western Blotting experiments. Normally, in previous SDS-PAGE step, the proteins are associated with enough SDS to transfer effectively from the gel to the membrane. However, when it comes to proteins tendency to precipitate or large proteins, SDS is necessary to the Western Blot transfer buffer.
The balance of SDS and methanol in the transfer buffer, protein size, and gel percentage are the main factors that affect transfer efficiency. About the current and transfer time for western blot, it's critical to choose the appropriate current and transfer time for a successful western blotting. Too low current or/and transfer time will lead to incomplete transfer; if the current or/and transfer time is too high, on the contrary, the proteins may migrate through the membrane too fast without being absorbed, and this case is especially for those small molecular weight proteins.
|Molecular weight (kDa)||Transfer time and current|
|< 20||1 Amp constant current for 45 mins or equivalent (250 mAmp for 3 hours or 500 mAmp for 90 mins)|
|20 -120||1 Amp constant current for 1 hour or equivalent (250 mAmp for 4 hours or 500 mAmp for 2 hours)|
|120 - 250||1 Amp constant current for 90 mins or equivalent (250 mAmp for 6 hours or 500 mAmp for 3 hours)|
|> 250||1 Amp constant current for 90 mins or equivalent (500 mAmp for 3.5 hours)|
|1||Be sure to run your samples in a low-concentration gel, 8% or less. Adding SDS to a final concentration of 0.1% in the transfer buffer will decrease the tendency of protein precipitation in the gel.|
|2||Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation.|
|3||Methanol is only necessary when nitrocellulose is used. If using PVDF, methanol can be removed from the transfer buffer altogether, and you just need to activate the PVDF with methanol before assembling the gel/membrane "sandwich" according to the right order.|
|4||Choose western blot wet transfer overnight at 4 ℃ instead of western blot semi-dry transfer.|
|1||Consider removing SDS from the transfer buffer and keep the methanol concentration at 20%.|
|2||Chicken antibodies tend to bind PVDF and other nylon-based membranes, leading to high background. Switching to a nitrocellulose membrane should help reduce background staining.|