Biochemistry Chapter 3 Outline Questions

Describe: antibody, antigen, antigenic determinant or epitope, immunoglobulin G.

- Antibody: synthesized by organisms in response to an antigen. - Antigen: foreign substance being targeted. - Epitope (antigenic determinant): a binding location on the antigen peptide. - immunoglobulin G: are antibodies or glycoprotein molecules produced by plasma cells.

Define and distinguish between the genome and the proteome.

- Genome is DNA - Proteome is the entire set of functional protein units of a cell that differs across cell types.

Describe how a quantitative enzyme assay can be used to calculate its specific activity during protein purification.

Protein activity is measured by examining how much Iight is absorbed by the solution. The purity is measured by determining the specific activity (enzyme activity / amount of enzyme).

Understand what does the sedimentation coefficient (S) "tells" you about a molecule.

The rate of movement of particles in a centrifuge. - The higher the mass, the greater the sedimentation speed. - If a protein is spherical in shape, it will move faster. - If a protein is more dense, it will move faster.

What is affinity chromatography?

Used to purify and separate crude mixtures of proteins based on their affinities to certain molecules attached to insoluble gel beads. Ex: group of interest (glucose) is attached to the beads. Proteins with the highest affinity for glucose will bind, while those with less affinity will travel to the bottom of the column.

Describe and compare indirect vs sandwich ELISA.

- Indirect ELISA: a method used to detect the presence of a specific antibody. The well is covered with specific antigens, the mixture of antibodies is added to the well & will bind to antigen if present. Enzyme-linked antibodies (2ndary antibodies) bind to the primary antibodies, if present. Unbound antibodies are washed off and a substrate specific to the enzyme is added. If present, the substrate and enzyme react, causing a color change in the solution. - Sandwich ELISA: a method used to detect the presence of an antigen. The antibody specific to the antigen is attached to the well. A sample containing the antigen is added, and if present, will bind to the antibody. An enzyme-linked monoclonal antibody is added, and it binds to the antigen bound to the antibody. The well is washed off and the substrate is added to react with enzyme. If present, the product will change color.

Contrast polyclonal and monoclonal antibodies and their preparation.

- Polyclonal antibodies are made using several different immune cells. They will have the affinity for the same antigen but different epitopes. - Monoclonal antibodies are made using identical immune cells that are all clones of a specific parent cell.

Compare Edman degradation with tandem mass spectrometry.

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Describe MALDI and TOF and explain, in general terms, how the techniques work together to allow application of mass spectrometry to proteins.

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Explain the application of overlap peptides to protein sequencing.

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Why is it necessary to cleave large proteins before determining their amino acid sequence?

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Describe how the use of fluorescent markers allows direct observation of changes within living cells.

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Briefly describe gradient centrifugation.

A density gradient is formed in a centrifuge tube and a mixture of proteins in solution is placed on top of the gradient. After centrifugation finishes, a small hole is made at the bottom of the tube and portions of the gradients are collected. Each gradient portion will have different proteins.

Describe Western blotting.

A method that utilizes antibodies to detect the presence of a protein in some sample. A monoclonal antibody, specific for the protein of interest, is manufacture in a lab. The sample of protein is exposed to SDS-polyacrylamide gel electrophoresis. The proteins are separated on the basis of size, where the smaller proteins migrate further down the gel. The separated protein sample is transferred onto a polymer sheet. The monoclonal antibody is added onto the polymer sheet containing the separate protein and binds to specific antigen of interest. A second, radioactively labeled, monoclonal antibody is created to bind to primary monoclonal antibody

Describe isoelectric focusing as a separation method.

A mixture of proteins are purified based on their isoelectric points.

What is salting out?

Adding a saturated salt solution to an aqueous layer, which decreases the solubility of the protein until they become insoluble and precipitate.

Describe how differential centrifugation would be used to produce a protein mixture from a cell homogenate.

In every centrifugation, the larger/heavier components (pellet) move to the bottom of the test tube. Smaller/lighter components (supernatant) remains at the top.

What is the isoelectric point (pI) of a protein? Of an amino acid?

Isoelectric point (pI) is the pH value at which the protein as a net charge of zero.

Explain the determination of protein mass by SDS-PAGE.

Larger proteins move slowly due to friction compared to smaller proteins. That is why larger proteins will be higher along the side.

What is ion exchange chromatography?

Purifies a mixture of proteins based on their net charge. The column is filled with charged gel beads. - Anion Exchange (Positive) beads catch negatively charged amino acids. Elute first: positive proteins. Elute last: negative proteins. - Cation Exchange (Negative) beads catch positively charged amino acids. Elute first: negative proteins. Elute last: positive proteins.

What is gel filtration chromatography? Aka molecular exclusion chromatography.

Separates protein based on their size. - Smaller proteins enter porous beads (emerge last). - Larger ones cannot enter, thus they reach the bottom first.

What is dialysis?

Separates proteins from smaller molecules and ions.

What is high-performance liquid chromatography?

Separates proteins on the basis of polarity. The solvent or mobile phase is forced through under high pressures, making the elution process faster.

Describe the principle of electrophoresis and its application in the separation of proteins.

Separation of proteins based on size. Proteins are denatured in a denaturing solution. SDS anions attach onto the side chains of amino acids, making them negative. As the SDS complexes move down the gel, they are dragged by the electric field, moving down.