Why is blotting done

The fragments are separated by size on an agarose or polyacrylamide gel via electrophoresis. Smaller fragments will migrate farther on the gel than larger ones. Following electrophoresis, the DNA on the gel is transferred to a nylon membrane.

The membrane is incubated with a nucleic acid probe that has a sequence homologous to the target sequence and is labeled with radioactivity, fluorescent dye, or an enzyme capable of generating a chemiluminescent signal.

Hybridization of complementary sequences occurs during incubation, and the unhybridized probe is removed by washing with buffer. The fully hybridized labeled probe molecules will remain bound to the blot. Detection methods differ based on the probe label; radiolabeled probes are visualized with X-ray film or phosphorimaging, and enzymatically labeled probes are visualized with chemiluminescent substrate.

Northern blots are used to determine the identity, size, and abundance of specific RNA sequences. Large RNAs are separated by electrophoresis on a formaldehyde agarose gel or glyoxal agarose gel, which prevents normal base paring and maintains RNA in a denatured state. This gel allows small molecules to move faster than bigger ones. The separated molecules are then pressed against a membrane, which helps move the molecules from the gel onto the membrane.

The molecules stick to the membrane, but stay in the same location, apart from each other, as if they were still in the gel. Western blotting is a common technique for separating proteins by size, but in straight columns. These parallel columns allow researchers to compare the amount of a protein across different samples that are run right next to each other, like bowling lanes.

For example, if you were testing the effect of different amounts of a drug on cell growth, you would treat four different groups of cells with a different amount of drug. Then you could break the cells open and run the proteins of each group in separate lanes on a gel. Spreading the proteins out in this way allows you to see what an increasing concentration of drugs does to a certain protein.

Northern blotting is used to detect RNA. Cells can be broken open to release their RNA. The RNA from different cell types can be run on separate lanes on a gel. The gel spreads the different RNA by size. This method allows a researcher to determine if cells from a certain disease have more of this RNA or less of that RNA.

Northern blotting may reveal how a disease is working at the level of RNA production. A restriction enzyme is used to cut a sample of DNA into fragments that are separated using gel electrophoresis. The DNA fragments are transferred out of the gel to the surface of a membrane. The membrane is exposed to a DNA probe labeled with a radioactive or chemical tag.

If the probe binds to the membrane, then the probe sequence is present in the sample. A Southern blot is a way to analyze DNA molecules.