Western Dot blot

Dot blotting is a method of applying proteins directly onto a membrane. A dissolved sample is pulled through the membrane by either applying a vacuum, absorption, or intrusion; proteins bind to the membrane and the other sample components pass through. The proteins on the membrane are then available for analysis. This technique can be used either as a qualitative method for the rapid screening of a large number of samples or as a quantitative technique. It is especially useful for testing the suitability of experimental design parameters.

When preparing blots by filtration, keep in mind the following:

• Detergents can inhibit the binding of proteins to the membrane. Buffers used for sample dissolution and washing should contain no more than 0.05% detergent if required. For Sample Buffer (0.4%SDS) dilute 1:7 with the buffer to achieve 0.05% SDS.
• Choose a sample volume large enough to cover the exposed membrane in each well, but be careful not to exceed the binding capacity of the membrane. In a blotting unit, the binding capacity of Immobilon-P is typically 5 to 10 mg/well, depending on the particular protein, the surface area of the exposed membrane, the filtration rate, and the buffer formulation.
• High particulate loads or viscosity will reduce the flow rate and clog the membrane. Centrifuge samples with particulates, and apply only the supernatant to the membrane. Dilute viscous samples in the buffer.

Manual Spotting Methods

Required Equipment and Solutions

• Two sheets of Immobilon-P membrane cut to size for blotting unit
• Filter paper, cut to size for blotting unit (i.e. WhatmanTM 3MM filter paper)
• Methanol, 100%
• Ultrapure water (produced by the Milli-Q® System)
• Buffer, for sample loading and wash
• The blotting unit, dot blot or slot blot format

Set-Up

1. Prepare the Immobilon-P membrane: 

• Wet the membrane by laying it on the surface of methanol for 15 seconds. Do not immerse. The membrane should uniformly change from opaque to semi-transparent.
• Carefully place the membrane in ultrapure water and soak for 2 minutes.
• Carefully place the membrane in the buffer and let equilibrate for at least 5 minutes.
• Dissolve the sample in buffer.
• If the sample solution is cloudy, centrifuge to remove particles.
• If the sample is viscous, dilute with additional buffer.

Stack

• Assemble stack as follows (from the bottom up):
• Place paper towels on the work surface (bottom towels should remain dry throughout the blotting procedure).
• Place dry filter paper (i.e. Whatman 3MM paper) on paper towels.
• Place filter paper (prewet with buffer) on dry filter paper.
• Place prewet Immobilon-P membrane on wet filter paper.
• Spot 1 – 5 mL of sample onto the membrane. The sample should wick into the membrane. The membrane should be wet enough to absorb the sample, but not so wet that the sample spreads across the membrane.
• After sample is absorbed, place membrane on clean filter paper to dry