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Blotting - The Procedure

gel paper nitrocellulose molecules

All blotting procedures begin with a standard process called gel electrophoresis. During this step, DNA, RNA, or proteins are loaded on to an agarose or acrylamide gel (that functions like a molecular sieve) and are then run through an electric field. Two types of gels are commonly used: agarose gels and acrylamide gels. Agarose gels are based on a meshwork of agar filaments and are most often used to analyze DNA and RNA. Acrylamide gels are based on a meshwork formed from the chemical acrylamide and used most often to analyze proteins. Gels are loaded with a mixture of many differently sized molecules. When pulled through the gel by the electric current, they will separate into separate pools on the basis of their size; smaller molecules migrate farther through the gel than larger molecules. These separate pools of molecules will appear as bands on the gel if they are stained with an appropriate dye. After the molecules have been fractionated on the gel, they are ready for transfer to the nitrocellulose paper.

Transfer is initiated when the gel is retrieved from the electrophoresis apparatus and the nitrocellulose paper is carefully laid on top of the gel. The objective now is to transfer the bands of molecules found in the gel over to the nitrocellulose paper. Here they become immobilized, and will reflect the pattern seen on the gel. The paper now serves as a type of permanent record of the gel's banding pattern that can be used for further analysis.

There are two basic ways the actual transfer, or blotting, is carried out. One method takes a "sandwich" of gel and nitrocellulose paper and places it in a special apparatus that sets up an electric field running perpendicular to the band as preserved in the gel. This pulls the bands of molecules out of the gel, and they are immediately absorbed onto the nitrocellulose paper. This method is most commonly employed in Western (protein) blots.

The other method, commonly employed with Southern and Northern blots, lays the gel on top of a platform that in turn is placed in a tray containing a buffer solution. Underneath the gel is a strip of blotting paper that is folded down on each side of the platform, so that it dips into the buffer to serve as a wick. On top of the gel are placed, first, the strip of nitrocellulose paper, then several pieces of blotting paper, and finally a small stack of paper towels. A weight is then placed on top of the paper towels. The buffer flows up the blotting paper "wick" by capillary action, then through the gel, through the nitrocellulose paper, and ultimately into the paper towels. The DNA or RNA in the gel moves with the buffer but sticks to the nitrocellulose paper on contact. The paper towels soak up the transfer buffer, but only after it has passed through the gel and deposited the DNA or RNA on the nitrocellulose paper.

After transfer has been completed, the nitrocellulose paper can be examined by using probes. Short fragments of DNA that have a nucleotide sequence complementary to the molecule being analyzed are normally used as probes in Southern and Northern blots. Antibodies that react with the protein being analyzed are used as probes in a Western blot. In either case, the probe is "labeled," usually by making it radioactive, so that it is easy to identify. In all blotting experiments, the nitrocellulose paper is placed in a chamber full of buffer and mixed with the probe, which then binds to the molecule that is being studied. This is called the hybridization step. Detection of the probe indirectly detects the molecules being studied.

Blotting - Illustrative Examples [next]

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