Dot/Slot (Filtration) Blotting

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.

Dot/Slot (Filtration) blotting topics: possible causes and remedies:

• Slow or No Filtration of the Sample Through the Membrane
• Little or No Protein Observed on the Blot
• Stained Blot Is Not Uniform
• Protein Smeared Across the Top of the Membrane
• Protein Smeared Across the Back of the Membrane

Figure 1. Detection of transferrin on a dot blot of human serum using Immobilon® Western Chemiluminescent HRP Substrate.

Slow or No Filtration of the Sample Through the Membrane
Possible Cause	Remedy
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.

Little or No Protein Observed on the Blot
Possible Cause	Remedy
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.

Stained Blot Is Not Uniform
Possible Cause	Remedy
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.

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

Protein Smeared Across the Back of the Membrane
Possible Cause	Remedy
Membrane capacity was exceeded	Reduce the amount of protein loaded into the well.