Western Blot Transfer Efficiency – The Good, the Bad, and the Ugly.
Protein visualization is key to a successful Western blot.
by Amy Archuleta
by Amy Archuleta
On our tech tips page, we advocate using Ponceau S stain to illustrate the efficiency of transfer from gel to membrane after removing it from the transfer apparatus.
Ponceau S staining is a wonderful way of illustrating the transfer efficiency as a whole, but also for highlighting any issues with small sections of the blot that could be problematic for that small portion, but that don’t negatively impact your entire blot.
These abnormalities (or normalities if your technique needs improvement) are invisible on a blot with no Ponceau S stain but become terrifyingly obvious after staining. They can happen in both semi-dry and tank (wet) transfer systems.
At right are some examples from a tank transfer set-up. The majority of the rat caudate lysate blot at right illustrates a beautiful transfer for mid-range MWs. There are two tiny bubbles on the far left, but these are easily avoided. A strange smudge near the bottom right, and some inconsistent spotting in the highest MWs illustrate that these sections of the blot are not ideal for bands that would show up in these specific areas.
Bubbling can happen for many reasons. A bubble appears as a white spot on your membrane in the middle of the field of red Ponceau S stain. Bubbles are the result of air being trapped between the membrane and the gel during transfer. Usually bubbling is minor, and if it is just a couple of bubbles like in the blot above, then I simply use a gel pen to circle them on the blot. Then when I use the blot later and the Ponceau S is washed away during blocking and incubation steps, I will still be able to see the compromised portion of the blot. This allows me to use the rest of the blot without worry.
Worst Case Scenario: Pictured below are blots of rat testes lysate run at the same time, in the same transfer apparatus, where the bubbling rendered the blots impossible to use. Actually, bubbling is only one of MANY issues on these blots! The left blot shows the bubbling as it appeared when emerging from Ponceau S stain. The right shows a similar blot on which I have circled all of the incriminating bubbles. Circling makes it very obvious that virtually no portion of this blot is usable.
All three of these issues are very common. Most of them can be solved by tightly packing the transfer sandwich.
This type of irregularity can appear differently (as seen in the image below) but is always indicative of non-uniform transfer of the protein in the gel to the membrane. The transfer sandwich must apply equal and firm pressure across the entire blot. Protein transfer efficiency will reflect any differences in pressure.
If the transfer sandwich has inconsistent pressure across the height of the membrane, horizontal waviness can appear. In the blot at right, it is clear that the gel was not tightly pressed against the membrane during transfer, allowing for buffer to pass between the two. At the top of the membrane, this resulted in the transferring protein “swimming” from one to the other and not maintaining its exact position. At the bottom of the membrane, what appears to be the edge of the gel is wavy and it is clear that swimming protein deposited itself beneath the gel’s edge.
Unsurprisingly, transfer sandwiches with insufficient pressure between the gel and the membrane can result in a loss of sharpness in the banding pattern. It is important to note that different lysates and different lysate preparations can also affect the banding pattern. However, it is always a good idea to try and minimize the smudge effect that can be caused by a too-loose transfer sandwich.
One of the main reasons that we transfer proteins from gels to membranes before probing with antibody is that gels are so fragile. When removing a gel from between the glass plates, placing it in the transfer apparatus, and assembling the sandwich, it is very easy to tear the gel. This doesn’t have to spoil your transfer, as the rest of the blot is probably fine. However once again it is vital to stain your membrane with Ponceau S to visualize and mark the problem area.
If you are sure that the above culprits aren’t the issue, check the function of your transfer apparatus and power source or your buffers.
There are a daunting number of variables that can affect a Western blot experiment. Tissue/cell lysis and preparation is a key component. Read about lysis buffers here, and goopy lysate here. SDS-PAGE efficiency is another hurdle. Read the basic science of SDS-PAGE here. After transfer to a membrane, antibodies can prove problematic for a variety of reasons. Read about antibody variability and validation here and here. Read about issues with secondary antibodies here. Read about the VERY basics of Western blotting here.
With all of these possible pitfalls, transfer efficiency is one issue that can usually be addressed quickly by three steps: