Protein Assays Flashcards
Gel electrophoresis
Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. When a mixture of proteins is applied to a gel and an electric current is ap- plied, smaller proteins migrate faster through the gel than do larger proteins.
What is centrifugation used for?
Centrifugation is used for two basic purposes: (1) as a preparative technique to separate one type of material from others and (2) as an analytical technique to measure physi- cal properties (e.g., molecular weight, density, shape, and equilibrium binding constants) of macromolecules. The sedimentation constant, s, of a protein is a measure of its sedimentation rate.
Types of liquid chromatography
- gel filtration chromatography
- ion exchange chromatography
- affinity chromatography
What is liquid chromatography?
In this technique, called liquid chromatography, the sample is placed on top of a tightly packed column of spherical beads held within a glass cylinder. The nature of these beads determines whether the separation of proteins depends on differences in mass, charge, or binding affinity.
Gel filtration chromatography
Proteins that differ in mass can be separated on a column composed of porous beads made from polyacrylamide, dextran (a bacterial polysaccha- ride), or agarose (a seaweed derivative).
Although proteins flow around the spherical beads in gel filtration chromatography, they spend some time within the large depressions that cover a bead’s sur- face. Because smaller proteins can penetrate into these depressions more easily than can larger proteins, they travel through a gel filtration column more slowly than do larger proteins). (In contrast, proteins migrate through the pores in an electrophoretic gel; thus smaller proteins move faster than larger ones.)
Ion exchange chromatography
proteins are separated on the basis of differences in their charges. This technique makes use of specially modified beads whose surfaces are covered by amino groups or car- boxyl groups and thus carry either a positive charge (NH3) or a negative charge (COO) at neutral pH
Affinity chromatography
In this technique, ligand molecules that bind to the protein of interest are covalently attached to the beads used to form the column. Ligands can be enzyme substrates or other small molecules that bind to specific proteins. In a widely used form of this technique, antibody-affinity chro- matography, the attached ligand is an antibody specific for the desired protein
An important pathway for protein degragation
Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into shorter amino acid sequences and used in synthesizing new proteins. Proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a polyubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein.
Using enzymes and antibodies to detect specific proteins
Chromogenic and Light-Emitting Enzyme Reactions
- enzyme assays are based on the ability to detect the loss of substrate or the formation of product. Some enzyme assays utilize chromogenic substrates, which change color in the course of the reaction
- luciferase, an en- zyme present in fireflies and some bacteria, can be linked to an antibody. In the presence of ATP and luciferin, luciferase catalyzes a light-emitting reaction
Western blotting
A powerful method for detecting a par- ticular protein in a complex mixture combines the superior resolving power of gel electrophoresis, the specificity of an- tibodies, and the sensitivity of enzyme assays. Called Western blotting, or immunoblotting, this multistep procedure is commonly used to separate proteins and then identify a spe- cific protein of interest. As shown in Figure 3-35, two dif- ferent antibodies are used in this method, one specific for the desired protein and the other linked to a reporter enzyme.
Steps in Western Blotting
Step 1 : After a protein mixture has been electrophoresed through an SDS gel, the separated bands are transferred (blotted) from the gel onto a porous membrane.
Step 2 : The membrane is flooded with a solution of antibody (Ab1) specific for the desired protein. Only the band containing this protein binds the antibody, forming a layer of antibody molecules (although their positioncannot be seen at this point). After sufficient time for binding, the membrane is washed to remove unbound Ab1.
Step 3 : The membrane is incubated with a second antibody (Ab2) that binds to the bound Ab1. This second antibody is covalently linked to alkaline phosphatase, which catalyzes a chromogenic reaction.
Step 4 : Finally, the substrate is added and a deep purple precipitate forms, marking the band containing the desired protein.
Edman degradation
The classic method for determining the amino acid sequence of a protein is Edman degradation. In this procedure, the free amino group of the N-terminal amino acid of a polypeptide is labeled, and the labeled amino acid is then cleaved from the polypeptide and identified by high-pressure liquid chro- matography. The polypeptide is left one residue shorter, with a new amino acid at the N-terminus. The cycle is repeated on the ever shortening polypeptide until all the residues have been identified.
X-ray christallography
In this technique, beams of x-rays are passed through a protein crystal in which millions of protein molecules are precisely aligned with one another in a rigid array characteristic of the protein. The wavelengths of x-rays are about 0.1–0.2 nm, short enough to resolve the atoms in the protein crystal. Atoms in the crystal scatter the x-rays, which produce a dif- fraction pattern of discrete spots when they are intercepted by photographic film (Figure 3-38). Such patterns are ex- tremely complex—composed of as many as 25,000 diffrac- tion spots for a small protein. Elaborate calculations and modifications of the protein (such as the binding of heavy metals) must be made to interpret the diffraction pattern and to solve the structure of the protein. The process is analogous to reconstructing the precise shape of a rock from the rip- ples that it creates in a pond. To date, the detailed three- dimensional structures of more than 10,000 proteins have been established by x-ray crystallography.
