2.3b Content Tutorial
Introduction to Blotting Techniques:
Blotting is the method of putting DNA, RNA or protein onto a membrane for further studies and detection. Usually prior to blotting the molecules are separated based on size or mass by electrophoresis (Module Subset II-a.). Like total protein electrophoresis, DNA and RNA can be separated by size/mass, but charge is irrelevant (DNA and RNA are negatively charged). In protein electrophoresis for molecular biology, proteins are separated by mass as charge is made negative by the pH of the buffer used. As there are no issues with charge, the only difference with the molecules position in the matrix following electrophoresis is the length of the sequence. Thus it is possible to determine the length of a piece of DNA or RNA or mass of a protein by its position in a gel relative to a standard marker. This type of analysis is important in epidemiology, forensics and in just checking up on your PCR reaction. For proteins many techniques are available to determine function as well as mass.
Two major types of matrix are used for electrophoresis; agarose gel or acrylamide gel. Agarose is of superior optical clarity compared to microbiology agar, but is handled in similar ways. Acrylamide is a crosslinked polymer that enables the use of small amounts of sample, separation of very similar pieces (sequencing gels for example) and are able to take a great deal of heat during the electrophoresis run. Buffers are added to the gels when they are made. DNA and RNA can be visualized in the gels after electrophoresis by the addition of a fluorescent dye that can be seen under UV light. Usually pictures or digital images are used for determination of the size of the bands in the gel.
The first blotting technique described was the Southern blot; published in 1975 by E. M. Southern. He described a technique for detecting specific DNA fragments after electrophoresis so a specific gene could be isolated from a complex DNA mixture. Since then, electrophoresis/blotting has been done with RNA (northern blots) and proteins (western blots). There has been lots of speculation on how you would run an eastern, but no one has solved it to date. [Note that the only technique that earns a capital initial is Southern!] Blots done without electrophoresis are called dot assays or dot blots or slot blots if a linear slot was used – often with filter paper migration of the target towards the probe. All blotting assays can be qualitative or quantitative depending on the fine details of the protocol. Detection of the DNA and RNA bands on the membrane is accomplished by the incorporation of some type of label in a specific probe. Probes will be described below. Western blots and protein dot blots are detected using specific antibodies, antibody conjugates and substrates. Western blots differ from DNA/RNA blots in that electrophoresis is used to accomplish the transfer of the proteins from the acrylamide gel to the membrane. Western blotting will be explored in detail in Module III, Protein Techniques.
In Southern/northern blotting, the DNA fragments/RNA molecules are separated according to size by gel electrophoresis in agarose gel. The technique is identical for RNA from DNA except for the reagents used and the sampling precautions of working with RNA (Module 2). Northern gels contain formamide – a potent inhibitor of RNases, so your sample is safe once it is in the gel. The next step in the Southern/northern blot technique is to transfer the bands of DNA to an inert membrane. When the membrane is being prepared for testing with a DNA probe, the DNA must be denatured to single stranded DNA before being fixed to the membrane. This may be accomplished by soaking the gel in sodium hydroxide or denaturing the DNA after it is blotted on the inert membrane.
Nitrocellulose or positively-charged nylon filters are used for blotting. The agarose gel is placed between several sheets of filter paper soaked in buffer, the nitrocellulose filter sits on top of this and paper towels sit on top of the nitrocellulose. As the buffer is drawn thought the agarose gel, it carries the DNA to the nitrocellulose or nylon filter, where it sticks. The blotting procedure may be accelerated by transmitting a vacuum through the gel and filter paper (vacuum blotting). Once the DNA is transferred to the filter, the DNA is permanently fixed the membrane if 1) it is the right kind of charged membrane, 2) the filter is heated several hours in a vacuum oven or seconds in a microwave, or 3) crosslinked by UV light (special UV linkers or your gel transilluminator).
The filter is then bathed with a solution containing first a blocking agent to prevent non-specific attachment of the detection probe to the membrane (pre-hyb), then the probe (hyb mix). The probe will hybridize to complementary DNA while any unbound probe will be washed off. Hybridization and detection of bound probe is described in much more detail in the sections below. In general, this is how a blotting protocol works.
University of Calgary Biotechnology Training Centre
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