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

PVDF coated areas

Areas of PVDF coated
with transferred protein
are capable of wetting
out in 20% methanol
while the surrounding
areas of PVDF are not.
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

High Background Staining

Possible Cause                               Remedy

Nonspecific protein binding to
the membrane