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 | |
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Possible Cause | Remedy |
Inappropriate membrane |
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Membrane wasn’t completely dried prior to wetting with methanol |
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Blot saturated with water only |
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Insufficiently saturated blot |
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Weak or Uneven Stain | |
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Possible Cause | Remedy |
Membrane wasn’t wetted in methanol prior to staining |
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Uneven/Splotchy Results | |
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Possible Cause | Remedy |
Use sufficient volume of incubation solutions and ensure that the membrane is completely covered with these solutions during incubation. |
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Air bubbles |
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Poor reagent quality |
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High Background Staining | |
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Possible Cause | Remedy |
Nonspecific protein binding to the membrane |
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Materials
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