There are several blot blunders that can mess with your transfer. Here's how to avoid them - or at least how to see they are there.
Contributed by Amy Archuleta
This is an abbreviated article. Read my full-length piece in our website’s Education section.
Protein Visualization is Key to a Successful Western Blot
Ponceau S staining is a wonderful way of illustrating the transfer efficiency across the whole blot, but also for highlighting any issues with small sections that could be problematic for that specific area, but that don’t negatively impact your entire blot.
The rat brain blot below illustrates a beautiful transfer for mid-range MWs. A couple of bubbles and a small smudge can be easily avoided thanks to the stain’s visualization.
Air bubbles trapped between the gel and the membrane can leave white circles on your blot.
After staining with Ponceau S, use a gel pen to outline these bubbles on your blot. This will allow you to visualize the location of the bubbles once the Ponceau S is washed away in the incubation steps. You can minimize bubbling be degassing your buffers, avoiding bubbling creation by pouring the buffer slowly when filling the tank, and carefully assembling your transfer sandwich by prewetting your sandwich pads and using a roller at each step to press out any trapped bubbles in the sandwich.
Tightly Packing the Transfer Sandwich can help avoid 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. The transfer sandwich must apply equal and firm pressure across the entire blot. Protein transfer efficiency will reflect any differences in pressure. Transfer sandwiches with insufficient pressure between the gel and the membrane can also result in a loss of sharpness in the banding pattern.
Compression of the sponges/pads used in the transfer sandwich is often to blame when these types of banding inconsistencies appear. Monitor your pad thickness regularly and supplement with additional pads if necessary to make sure that gel and membrane are pressed firmly together.
These types of issues can also indicate a problem with your gel system. 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 cause poor resolution.
Gels are delicate. If one is damaged in handling, staining can help you avoid any compromised area on your blot.
One of the main reasons that we transfer proteins from gels to a membrane before probing with antibody is that gels are so fragile. Gels are notorious for sticking to the glass plates when removing them from the SDS-PAGE apparatus, so using a razor blade and squirt bottle of water to loosen the gel helps prevent damage.
As a last possibility, you may have transfer apparatus or buffer component issues.
If you are sure that the above options aren’t the issue, check the function of your transfer apparatus and power source or your buffers. Make sure that your electrodes are in good condition, are in the proper orientation, and that your power source is operational. Buffers can also cause problems, so make sure you are using the correct ones for your system / membranes. Buffers on occasion get contaminated or made incorrectly. Sometimes making fresh buffer can make a big difference.
3 take-homes for better blotting: bubble prevention, tight transfer sandwiches, and dye visualization.
There are a daunting number of variables that can affect a Western blot experiment. Lysate/protein/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.