Protein Analytical techniques

Question 1

Salting out

Answer 1

1. A protein purification technique
2. based on the fact that the solubility of most proteins is lowered at higher salt concentrations.
3. Consequently, different proteins will precipitate at varying salt concentrations.

Question 2

dialysis

Answer 2

1. The process of removing small molecules from a solution containing a mixture of large molecules and small molecules.
2. The mixture is placed in a bag made of a semipermeable membrane,
3. Is then placed in a different solution.
4. The membrane allows escape by the small molecules but not the large molecules.

Question 3

gel-filtration chromatography

Answer 3

1. A separation technique based on size differences.
2. A sample is applied to a column consisting of porous beads.
3. Large molecules move through the column faster because they cannot enter the beads and, thus, have a shorter path to travel.

Question 4

ion-exchange chromatography

Answer 4

1. A protein purification technique that relies on the charge of proteins.
2. Proteins are applied to an inert matrix to which is attached a charged moiety (e.g., a carboxylate group).
3. Proteins will bind to the matrix with an affinity proportional to their content of the counterion (i.e., positive charges in the case of the carboxylate matrix).

Question 5

cation exchange

Answer 5

1. Ion-exchange chromatography
2. A protein mixture is passed through a column containing a matrix bearing negative charges.
3. Proteins bearing positive charges will bind to the column
4. those with negative charges will pass through the column.

Question 6

anion exchange

Answer 6

1. Ion-exchange chromatography
2. A protein mixture is passed through a column containing a matrix bearing positive charges.
3. Proteins bearing negative charges will bind to the column
4. those with positive charges will pass through the column.

Question 7

affinity chromatography

Answer 7

1. A protein purification technique based on the high affinity many proteins have for specific chemical groups.
2. Such groups are attached to an inert matrix,
3. The protein sample is applied;
4. only those with an affinity for the groups will bind.

Question 8

high-performance liquid chromatography (HPLC)

Answer 8

1. A column chromatography technique in which the column materials are very finely divided
2. As a consequence, possess more interaction sites and thus greater resolving power.
3. Because the column is made of finer material, pressure must be applied to the column to obtain adequate flow rates.
4. The net result is both high resolution and rapid separation.

Question 9

gel electrophoresis

Answer 9

1. A technique used to separate charged molecules,
2. such as proteins and nucleic acids, which is
3. based on the fact that such molecules will move at differing rates through a gelatinous material,
4. such as polyacrylamide or agarose, when subjected to an electric field.
5. Separation depends on factors such as:
   
   1. net charge,
   2. size,
   3. shape of the molecules.

Question 10

isoelectric focusing

Answer 10

1. A technique for separating proteins.
2. A mixture of proteins is electrophoresed in a pH gradient;
3. each protein will migrate in the electrical field until it reaches its isoelectric point.

Question 11

two-dimensional electrophoresis

Answer 11

1. A means of analyzing a protein sample
2. in which the sample is initially fractionated in one dimension by isoelectric focussing,
3. and subsequently fractionated in a second dimension, perpendicular to the first, by SDS-polyacrylamide gel electrophoresis.

Question 12

enzyme-linked immunosorbent assay (ELISA)

Answer 12

1. An assay for quantifying the presence of an antigen
2. Uses an enzyme linked to an antibody to the antigen.

Question 13

western blotting

Answer 13

1. An immunoassay technique used to detect a specific protein in a cell or in body fluid.
2. A sample is electrophoresed in an SDS-polyacrylamide gel,
3. the resolved proteins are transferred to a polymer sheet,
4. Tthen an antibody specific for the protein of interest is incubated with the blotted sample;
5. other antibodies or radioactive markers may then be used to help visualize the desired antigen-antibody complex.

Question 14

fluorescence microscopy

Answer 14

1. An optical microscope capable viewing materials by reflection and absorption
2. as well as visualizing fluorescent materials.

Question 15

matrix-assisted laser desorption/ionization (MALDI)

Answer 15

1. A technique for determining a proteins mass.
2. The protein is evaporated to dryness in the presence of a volatile, aromatic compound that can absorb light at specific wavelengths.
3. A laser pulse excites and vaporizes the matrix, converting some of the protein into the gas phase.
4. Subsequent collisions enable the intermolecular transfer of charge, ionizing the protein.
5. The newly formed ions then enter the mass analyzer,
6. They are distinguished on the basis of their mass-to-charge ratios.

Question 16

electrospray ionization (ESI)

Answer 16

1. A mass spectrometry technique that allows the analysis of proteins.
2. A solution of the protein is passed through an electrically charged nozzle.
3. Droplets of the protein, now charged, emerge from the nozzle into a chamber of very low pressure,
4. evaporating the solvent and ultimately yielding the ionized protein.
5. The newly formed protein ions then enter the mass analyzer,
6. where they are distinguished on the basis of their mass-to-charge ratios.

Question 17

time-of-flight (TOF) mass analyzer

Answer 17

1. A mass analyzer used in conjunction with matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI).
2. The time-of-flight (TOF) analyzer accelerates the ions generated by MALDI or ESI through a chamber under a fixed electrostatic potential.
3. The mass of each ion can be determined by measuring the time required for each ion to pass through the chamber.

Question 18

Edman degradation

Answer 18

1. used in sequencing proteins.
2. Sequential removal of the N-terminal amino acid from a protein as a phenylthiohydantoin derivative;

Question 19

tandem mass spectrometry

Answer 19

1. The utilization of two mass analyzers to determine protein sequence.
2. Ions of proteins that have been analyzed by a mass spectrometer are broken into smaller peptide chains by bombardment with atoms of an inert gas such as helium or argon.
3. These new fragments can be passed through a second mass analyzer for further mass characterization.

Question 20

overlap peptide

Answer 20

1. Peptides resulting from degradation of a protein by two different procedures that are subsequently sequenced.
2. The sequence of a peptide from one degradation procedure frequently overlaps the sequences of two or more peptides of the other degradation procedure,
3. thereby establishing the order of the peptides.

Question 21

peptide mass fingerprinting

Answer 21

1. A technique for protein identification that involves cleaving a protein by chemical or enzymatic methods
2. followed by chromatographic separation and mass spectrometry.

Question 22

solid-phase method

Answer 22

1. A means of synthesizing discrete peptides
2. in which amino acids are added step-by-step to a growing peptide chain that is anchored to an insoluble matrix.

Question 23

x-ray crystallography

Answer 23

1. A technique to determine the three-dimensional structure of protein crystals at atomic resolution
2. by examining the diffraction pattern of x-rays striking the crystal.

Question 24

nuclear magnetic resonance (NMR) spectroscopy

Answer 24

1. Means of determining the structure of a protein in solution
2. based on the ability of certain atoms in a protein to absorb electromagnetic radiation.