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Dot/Slot (Filtration) Blotting

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Related Resources: Brochures | Application Notes
Detection of transferrin on a dot blot of human serum using Immobilon® Western Chemiluminescent HRP Substrate.
Detection of transferrin on a dot blot of human serum using Immobilon® Western Chemiluminescent HRP Substrate.
Dot blotting is an ideal technique for quickly assessing the levels of a target antigen across many samples at once. Also, it is a popular method for epitope mapping and screening antibodies for target specificity.

Click on the Dot/Slot (Filtration) blotting topics to read about the possible causes and remedies:

Slow or No Filtration of the Sample Through the Membrane

Possible CauseRemedy
Inadequate vacuum
  • Make sure the blotting unit is closed properly and the seal is intact.
  • Make sure the vacuum source (e.g. pump) is operating properly.
  • Seal off any open wells with a high quality laboratory tape. Increase vacuum level.
Membrane pores clogged
  • Centrifuge or filter samples to remove particulates.
  • Dilute viscous samples with buffer.
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Little or No Protein Observed on the Blot

Possible CauseRemedy
Not enough protein applied to the membrane
  • Minimize sample dilution and filter more sample through the membrane.
Detergents (e.g., SDS) may inhibit lower molecular weight proteins from binding to the membrane
  • Eliminate detergents if possible.
Stain not sensitive enough.
  • Use a more sensitive stain.
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Stained Blot Is Not Uniform

Possible CauseRemedy
Membrane structure was compressed by filter paper
  • Place a second membrane in the blotting unit to protect the membrane receiving the samples.
Air bubbles trapped in the interior of the membrane
  • Pre-wet membrane by laying it on the surface of the methanol. Immersing the membrane can entrap air.
Membrane not pre-wet in methanol
  • Membrane must be pre-wet with methanol; entire membrane should change uniformly from opaque to semi-transparent.
Air bubbles in the sample.
  • Carefully pipette samples into well to avoid the formation of air bubbles.
Not enough sample volume loaded
  • Sample must cover the entire exposed membrane area.
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Protein Smeared Across the Top of the Membrane

Possible CauseRemedy
Sample leaked across the wells
  • Make sure the blotting unit is properly assembled, closed and sealed prior to filtration.
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Protein Smeared Across the Back of the Membrane

Possible CauseRemedy
Membrane capacity was exceeded
  • Reduce the amount of protein loaded into the well.
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