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Protein Dot Blotting using Immobilon-P |
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 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.
Vacuum Filtration Method1
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.
• 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 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 buffer.
The following protocol describes a typical procedure for filtering proteins onto an Immobilon-P membrane.
Please review the instructions supplied with your blotting unit for additional information.
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
• Blotting unit, dot blot or slot blot format
* To ensure that the microporous structure of the membrane is not compressed when placed in the blotting unit, it is recommended that a second sheet of membrane be placed between the filter paper and the primary membrane.
Set Up
1. Prepare Immobilon-P membrane:
a. 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.
b. Carefully place the membrane in ultrapure water and soak for 2 minutes.
c. Carefully place the membrane in buffer and let equilibrate for at least 5 minutes.
2. Dissolve the sample in buffer.
a. If the sample solution is cloudy, centrifuge to remove particles.
b. If the sample is viscous, dilute with additional buffer.
Assemble the Blotting Unit
Note: See manufacturer's instructions for detailed assembly instructions.
1. Place one sheet of moistened filter paper on unit. (Note: Some units may require more than one sheet.) The filter paper in contact with the Immobilon-P membrane should be wet with the same buffer used to equilibrate the membrane.
2. Place two sheets of Immobilon-P membrane on top of the filter paper.
3. Close the unit.
4. Connect to vacuum line.
Filter
1. Briefly apply the vacuum to remove excess buffer. Please note that it is important that membrane remains wet while loading samples.
2. With the vacuum off, carefully pipette samples into the wells.
3. Apply vacuum to the blotting unit.
4. When all of the samples have filtered through the membrane, turn off the vacuum.
5. Add buffer to each well to wash down the sides; apply vacuum.
6. When all of the wash buffer has filtered through the membrane, turn off the vacuum.
Remove the Blot
1. Open the blotting unit.
2. Using forceps, carefully remove the membrane.
3. Place the membrane on clean filter paper to dry.
Note: Do not allow the membrane to dry out if analysis of the bound protein requires the native conformation or enzyme activity.
Manual Spotting Methods
Stack
1. Prepare the membrane the same as above in the Vacuum Filtration Method. (see “Set Up” Step 1 a – c)
2. Assemble stack as follows (from the bottom up):
Place paper towels on work surface (bottom towels should remain dry throughout 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.
3. Spot 1 – 5 mL of sample onto membrane. Sample should wick into membrane. Membrane should be wet enough to absorb sample, but not so wet that sample spreads across membrane.
4. After sample is absorbed, place membrane on clean filter paper to dry.
Dry (Intrusion, Pressure Blotting)2
1. Place a 1mL tuberculin syringe directly against dry Immobilon-P membrane and inject up to 50 mL of protein sample into membrane.
2. Place membrane on clean filter paper to dry.
References:
1. Protein Blotting Applications Guide, Technical Protocol TP001, 1997, Millipore Corporation, Bedford, Massachusetts, USA.
2. Oprandy, J.J., Olson, J.G., Scott, T.W. (1988). A rapid dot immunoassay for the detection of serum antibodies to Eastern equine encephalomyelitis and St. Louis encephalitis viruses in sentinel chickens. Am. J. Trop. Med. Hyg., 38 (1), 181-186.
Immobilon and Milli-Q are trademarks of Millipore Corporation or an affiliated company.
Whatman is a trademark of Whatman Paper Ltd.
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