Protein Visualization is Key to a Successful Western Blot
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.
Most Common Problem #1 – Bubbles
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.
- Recently mixed transfer buffer. The agitation of mixing transfer buffer causes bubbling that can then get trapped.
- Solution – Degas your transfer buffer. Let it sit for a while.
- Solution – Mix fresh buffer without shaking – use a stir bar.
- Pouring transfer buffer into transfer apparatus too quickly.
- Solution – Pour the transfer buffer slowly into your tank to keep bubbling at a minimum. This is similar to the beer-against-the-side-of-the-glass technique.
- Careless sandwich assembly. One of the most important things to do while setting up your transfer is to be observant and mindful as each layer is added. At each step in the process (filter paper – gel – membrane – filter paper) it is important to look for and remove any bubbles.
- Solution: Rollers. Bubbles can be minimized by using a rolling device on the top filter paper after sandwich assembly to apply additional pressure and squeeze out bubbles. These rollers come in many different sizes and shapes, and you can even use a conical tube or cut serological pipette.
- Solution: Pre-wetting transfer sandwich pads (sponges) with transfer buffer. If you are using a wet transfer box, adding dry pads on top of your submerged blot and filter paper can result in air sneaking into your sandwich. The pads act as a sponge, pulling buffer away from your blot.
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.
Most Common Problems #2,3,4: Vertical Variation / Horizontal Waves / Smudged Banding
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.
- Insufficient tightness in the transfer sandwich. This is usually due to compressed sandwich pads. Over time and over many transfers, these pads become compressed just as any sponge would. Even after only a few uses, the compression is obvious.
- Solution: Replace your sandwich pads frequently.
- Solution: Monitor the thickness of the pads and supplement your sandwich with additional filter paper (or an additional pad) if there is any pad compression.
- Issues with the SDS-gel. No membrane is going show discreet band separation if the gel from which the proteins were transferred had a blurred resolution to begin with.
- Solution: There are many things that can go wrong at this early step and are beyond the scope of this article. However, things to consider are: correct buffer concentrations and pHs, fresh components, and appropriate lysis buffer. If you are pouring your own gels, incomplete polymerization of your gel can also be to blame for poor resolution.
Common Problem #5 – Gel damage.
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.
- Stuck gel. Gels are notorious for sticking to the glass plates when removing them from the SDS-PAGE apparatus.
- Solution: Run a razor blade along the edges of your gel to separate it from the plate.
- Solution: Use a squeeze bottle with water to shoot liquid in between the gel and the plate to further loosen the gel before trying to remove it from the plate.
- Rough handling. Although they are relatively hardy, the truth remains that we’re dealing with thin, gelatinous sheets. Delicacy is the order of the day.
- Solution: Be more gentle.
Common Problem #6 – Transfer Apparatus or Buffer Component Issues.
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.
- Apparatus Issues. There could be a problem with your set-up that is keeping you from success.
- Solution: Black in back, red ahead! Make sure that your wires are attached to the correct electrode. If they are reversed, your proteins will be pushed away from the membrane and float away in the buffer.
- Solution: Damaged Electrodes. Check the coating on your anode for wear, tear, and scratches.
- Solution: Power Source. Is it still operational? Did you set it at the correct volts/amps?
- Buffer Weirdness. Depending on your membrane and proteins of interest, your buffer may be to blame.
- Solution: Reformulate your buffer. Research the buffer that works best in your system and with your membranes. Variables to address are methanol content, SDS content, salt concentration, and pH.
- Solution: Remake your buffer. Mistakes were made. It happens. Sometimes just making fresh buffer can solve a world of problems.
To Sum it Up
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:
- Taking time to prevent bubbles.
- Packing your sandwich tightly.
- Checking for transfer efficiency by staining with Ponceau S.