The ribosome is the cell's factory for protein synthesis. Each ribosome consists of two subunits, one large and one small, each of which is made up of RNA plus a large number of proteins. The ribosomal subunits and their RNAs are named using a parameter, called the S value. The S value, or Svedberg unit, is a sedimentation rate. It is a measure of how fast a molecule moves in a gravitational field. For example, the bigger a ribosomal subunit, the quicker it will sediment and the larger the S value. Prokaryotic ribosomes, and those found
IN DEPTH 8.1 How We Study Proteins in One Dimension_
The technique known as SDS-PAGE is widely used to analyze the spectrum of proteins made by a particular tissue, cell type, or organelle. It is also invaluable for assessing the purity of isolated proteins. SDS stands for sodium dodecyl sulfate and PAGE for polyacrylamide gel electrophoresis.
The aim of the technique is to denature the proteins to be analyzed and then to separate them according to their size in an electrical field. To do this we first add a chemical called 2-mercaptoethanol to the protein sample. This will break any disulfide bonds (page 191) within a protein or between protein subunits. Next SDS, which is an anionic detergent, is added and the protein sample boiled. SDS coats each protein chain with negative charge. Each individual polypeptide in the sample becomes covered with an overall net negative charge. This means that when placed in an electrical field the SDS-coated proteins will separate according to their size because the smaller proteins move most quickly toward the positive electrode or anode. Polyacrylamide provides the matrix through which the proteins move during electrophoresis. The monomeric form, acrylamide, is poured into a mold. A solid but porous gel forms as the acrylamide polymerizes. The shape of the mold is such that wells are formed in the gel into which the protein sample can be loaded for electrophoresis.
When electrophoresis is complete, the proteins are stained by incubating the gel in a solution of Coomassie brilliant blue. Each protein band stains blue and is detectable by eye. However, if the amount of protein is very low, a more sensitive detection system is needed such as a silver stain. Proteins of known molecular mass are also electrophoresed on the gel. By comparison with the standard proteins, the mass of an unknown protein can be determined.
If we want to follow the fate of a single protein in a complex mixture of proteins, we combine SDS-PAGE with a technique called western blotting. The name western blotting is, like northern blotting, a play on the name of Dr. Ed Southern who devised the technique of Southern blotting to analyze DNA (Table 7.2).
The protein mixture is separated by SDS-PAGE. A nylon membrane is then placed up against the polyacrylamide gel and picks up the proteins, so that the pattern of protein spots on the original polyacrylamide gel is preserved on the nylon membrane. The nylon membrane is then incubated with an antibody specific for the protein of interest. This antibody, the primary antibody, will seek out and bind to its partner protein on the nylon membrane. A second antibody is added that will bind to the primary antibody. To be able to detect the specific protein of interest on the membrane, the secondary antibody is attached to an enzyme. In the figure shown, the enzyme used was horseradish peroxidase. A substrate is added and is converted by the enzyme into a colored product. The protein of interest isseen as a colored band on the nylon membrane. The same enzyme-linked secondary antibody can be used in other laboratories or at other times for western blotting of many different proteins because the specificity is determined by the unlabeled primary antibody.
Part A of the figure shows the Coomassie brilliant-blue-stained pattern of proteins isolated from the endoplasmic reticulum of liver. The leftmost lane is from a phenobarbital-treated animal while the middle lane is from an untreated control animal. The dark bands indicate the presence of protein. The spectrum of proteins is very similar in the two samples, except that a band with a relative molecular mass (Mr) of about 52,000 is much darker in the sample from the treated animal. This tells us that drug treatment has caused an increase in the production of a protein with this relative molecular mass. Western blotting (part B) using an antiCYP2B1
Western blot (b)
Western blot (b)
antibody confirms that the induced protein is the cytochrome P450 protein known as CYP2B1. The CYP2B1 gene is activated by phenobarbital to produce more CYP2B1 protein to metabolize and clear the drug from the body (page 249).
We already showed how northern blotting revealed that transcription of the CYP2B1 gene is increased after phenobarbital treatment (Fig. 7.13 on Page 148). The western blot shown here demonstrates that, as expected, the amount of CYP2B1 protein is increased as well.
Sodium dodecyl sulfate (SDS) is a major constituent of hair shampoo, where it is usually called by its alternative name of sodium lauryl sulfate.
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