Tools of Protein Biochemistry

Question 1

General Electrophoresis

Answer 1

• charged molecules in solution move in an electrical field (+ toward - anode, vice versa).
• different charged molecules migrate in an electrical field at different rates depending on their total charge, size, and shape.
• can be native-PAGE or SDS-PAGE

Question 2

Nondenaturing Electrophoresis (Native-PAGE)

Answer 2

• proteins retain tertiary and quaternary structures
• rate of migration complex function of mass, shape, and charge

Question 3

Denaturing Electrophoresis (SDS-PAGE)

Answer 3

• Uses SDS (anionic detergent with hydrophobic body) and a reducing agent
• samples boiled (to denature proteins) and then ran on gel. 2°, 3°, and 4° conformations and disulfide bonds broken. Peptide bonds (1°) remain.
• SDS binds to protein backbone
• Migration proportional to mass
• effective way to determine molecular weight of protein, # of subunits in a purified protein, and to visualize many proteins simultaneously

Question 4

Main use of SDS-PAGE

Answer 4

effective way to determine molecular weight of protein, # of subunits in a purified protein, and to visualize many proteins simultaneously

Question 5

Main use of Western Blot

Answer 5

• identify specific protein of interest in a complex mixture of proteins

Question 6

Western Blotting (General)

Answer 6

• separating power of electrophoresis with the specificity of the antigen-antibody interaction
• uses antibodies to identify a specific protein
  
  • immunological method

Question 7

Western Blotting Steps

Answer 7

1. SDS-PAGE
2. Transfer proteins from gel to paper (via perpendicular electrical current)
3. Expose paper to a 1° antibody that recognizes the protein or an epitope placed in the protein sequence
4. Wash away unbound 1° antibodies
5. Expose paper to 2° antibody that can be detected via color or flourescence. 2° antibody binds to 1° antibody.

Question 8

Affinity Purification of Proteins (General)

Answer 8

• uses antibodies covelently linked to polysterene beads are mixed into protein mixture to allow the specific protein antigen to bind
• Beads/mixture washed, protein can then be eluted
• difficulty is that high affinity for protein-antibody interaction may require harsh (denaturing) conditions to elute protein. This yeilds inactive protein.

Question 9

Co-immunoprecipitation of Proteins (General)

Answer 9

• proteins that interact with one another will often remain bound to one another during purification in gentle conditions
• Only one of the proteins in the protein-protein interaction need the epitope recognized by the antibody
• once both proteins purified, they can be separated via SDS-PAGE to determine individual identities
• If antibodies to both interacting proteins are available, can use Western Blotting to determine relative amounts of each protein under different conditions

Question 10

Main Use of Co-immunoprecipitation of Proteins

Answer 10

• identify interacting proteins as well as to monitor changes in these interactions
• determine relative amounts of each interacting protein under different conditions using Western Blotting if antibodies are available for both interacting proteins

Question 11

Epitope Tagging (general)

Answer 11

• used to determine functions of proteins
• a recombinant fusion protein is generated, in which epitope is bound to C- or N-terminus of the coding region of the protein of interest
• proteins can be immunoprecipitated using antibodies against the epitope
• particularly useful for studying proteins encoded by newly identified genes

Question 12

Main Uses of Epitope-tagging

Answer 12

1. determining the size, intracellular localization, and abundance of proteins
2. monitoring post-translational modifications
3. studying receptor binding and internalization of exogenous proteins
4. establishing the identity of a protein within a protein complex

Question 13

Enzyme-linked immunosorbent assays (ELISAs) General

Answer 13

• plate-based assay designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones
• enzyme that reacts with a colorless substrate to produce a colored product is covalently linked to a specific antibody that recognizes a target antigen
• presence of colored product indicates presence of antigen
• two forms of ELISA (direct and indirect)

Question 14

Indirect versus Direct ELISA

Answer 14

• Direct ELISA has enzyme on 1° antibody. No 2° antibody needed.
• Indirect ELISA has enzyme on 2° antibody. 1° antibody binds to target antigen, 2° antibody with enzyme added and binds to 1° antibody, reaction to produce color occurs

Question 15

Immunohistochemistry (general)

Answer 15

• identifies specific tissue components by means of specific antigen/antibody reaction tagged with a visible label
• aids in visualizing the distribution and localization of specific cellular components within a cell or tissue
• provides in-situ information

Question 16

Major components in a complete immunohistochemistry experiment:

Answer 16

1. 1° antibody binds to specific antigen
2. antibody-antigen complex is formed by incubation with a 2° enzyme-conjugated antibody
3. Enzyme on 2° antibody catalyzes to generate colored deposits at the sites of 1° antibody-antigen binding

Question 17

Protein Microarrays General

Answer 17

• analytical versus functional protein microarrays
• contain small amounts of purified proteins

Question 18

Functional protein arrays are mainly used to study various types of protein activities, including:

Answer 18

1. protein-protein, protein-lipid, protein-DNA, protein-drug, and protein-peptide interactions
2. identify enzyme substrates
3. profile immune responses