Protein Visualization

Most Western blot users visualize transferred proteins using stains, such as Ponceau S or Coomassie^® Blue. However, users of PVDF membrane have the opportunity to assess transfer efficiency using transillumination. Areas of PVDF coated with transferred protein are capable of wetting out in 20% methanol while the surrounding areas of PVDF are not. In the areas where the PVDF wets, it becomes optically transparent, allowing visualization of protein bands using backlighting and photographic archiving.

Click on the protein visualization topics to read about the possible causes and remedies:

• Poor Detection by Transillumination
  
• Weak or Uneven Stain
  
• Uneven/Splotchy Results
  
• High Background Staining
  

Poor Detection by Transillumination

Possible Cause	Remedy
Inappropriate membrane	Transillumination works best with Immobilon®-P transfer membrane. It is not recommended for nitrocellulose or Immobilon®-PSQ transfer membrane.
Membrane wasn’t completely dried prior to wetting with methanol	Be sure that the membrane was dried completely after the transfer prior to immersing it in the 20% methanol solution. Make sure to use a 20% methanol solution.
Blot saturated with water only	Saturate the blot with 20% methanol.
Insufficiently saturated blot	Increase concentration of methanol up to 40%.

Weak or Uneven Stain

Possible Cause	Remedy
Membrane wasn’t wetted in methanol prior to staining	The membrane must be pre-wet with methanol; the entire membrane should change uniformly from opaque to semitransparent.

Uneven/Splotchy Results

Possible Cause	Remedy
Use sufficient volume of incubation solutions and ensure that the membrane is completely covered with these solutions during incubation.	The container used should be large enough to allow solution to move freely across the blot. Do not incubate more than one blot at a time in that same container. In addition, the protein side of the blot should be facing up so as not to be interacting with the bottom surface of the container.
Air bubbles	The blot should not have any air bubbles on the surface. Gently pull the membrane across the edge of the container to remove bubbles.
Poor reagent quality	All of the buffers and reagents should be fresh and free of particulates and contaminants. Filtration of buffers with Millex® syringe filters or Stericup®, Steritop® or Steriflip® filter units and centrifugation of antibody stocks may be required.

High Background Staining

Possible Cause	Remedy
Nonspecific protein binding to the membrane	Make sure to use clean electrotransfer equipment, components, high quality reagents, and Milli-Q® water.