module 3

Question 1

proteome definition

Answer 1

total protein content in a cell

Question 2

types of proteomics (study of proteins)

Answer 2

structural - analyzes structure of proteins being produced expressional - considers protein expression under certain conditions
interaction - how proteins interact with other molecules

Question 3

protein purification and its steps

Answer 3

isolating proteins from their source

steps: 1. cell fractionation 2. series of centrifugation using centripetal acceleration

Question 4

dialysis

Answer 4

protein solution is put into a bag, bathed in a buffer solution. Large molecules remain and small molecules go through pores.

Solvents, ions and buffer can diffuse easily across the semipermeable membrane, but larger molecules are unable to pass through the pores.

Question 5

salting out

Answer 5

purification technique for proteins based on differential solubility in salt solutions, causes protein to compete with salt. The protein is precipitated and less water molecules are able to interact with the protein. Causes proteins to form hydrophobic bonds with each other.

Involves ammonium sulfate and hydrophobic amino acids on exterior interaction with water.

Question 6

Effect of salting out

Answer 6

salt in small concentrations causes ammonium and sulfate to be separated. When concentration of salt is increased, protein solubility increases and ceases to interact with water.

Question 7

Salting out for different protein molecules

Answer 7

Different protein molecules precipitate at different concentrations of salt solution allowing for some proteins to precipitate while others interact with the water.

Question 8

Column chromatography

Answer 8

includes stationary phase, which retards the flow of the sample and the mobile phase, where the mixture to be separated moves

Question 9

gel filtration chromatography

Answer 9

separates biomolecules on basis of size, smaller proteins move faster than larger proteins

Question 10

non porous vs porous resins

Answer 10

non porous proteins move faster

Question 11

affinity chromatography

Answer 11

powerful column separation procedure based on specific binding of molecules to ligands

Question 12

proteases

Answer 12

enzymes that typically break peptide bonds by binding to specific amino acid sequences in a protein and catalyzing their hydrolysis .

Question 13

SDS function

Answer 13

reduces proteins so they assume a rod like shape, useful for gel electrophoresis

Question 14

merceptoethanol

Answer 14

sulfhydryl-containing reagent that becomes oxidized as they reduce disulfide bonds in other molecules

Question 15

isoelectric focusing

Answer 15

pH gradient established in tube containing an acrylamide gel matrix. An electric field is applied and proteins move according to their charge. When molecules reach their pI, they stop moving.

Question 16

2D gel electrophoresis

Answer 16

SDS page and isoelectric focusing are combined for proteomics, allowing all proteins of a cell/tissue to be studied simultaneously.

First step is isoelectric focusing, second step is use of SDS, which coats proteins with negative charge and denatures them, allowing SDS page to separate proteins exclusively according to molecular weight .I

Question 17

Histidine properties

Answer 17

flexible proteins has certain affinity to different ions, allowing for it to be used as a tag in recombinant proteins

His-tags are often used to identify expressing cells when producing recombinant proteins.

Question 18

ion exchange chromatography

Answer 18

separates substances on the basis of charge

involves cation exchange media (negatively charged groups on stationary phase trying to slow positive molecules) and anion exchange media (positively charged stationary phase trapping negative molecules)

Question 19

What is true about SDS page?

Answer 19

smaller proteins move faster.

Question 20

HPLC (high phase liquid chromatography)

Answer 20

chromatography technique that gives fast and clean purifications

Question 21

Electrophoresis definition

Answer 21

separates molecules on the basis of ratio of charge to size

Question 22

What do specific activity of enzyme, fold purification, and total activity mean in regard to protein purification

Answer 22

Total activity always decreases during purification. When a protein is purified, the specific activity will increase as a product of the fold purification. If the total activity is decreased too much and fold purification is not that high, the step is most likely not needed.

Question 23

Strategy to detect primary structure of protein

Answer 23

several samples of interest are prepared to identify individual amino acids, identify the N terminal amino acid, deduce the sequence of smaller peptides, and combine info from overlapping peptides used.

Question 24

trypsin

Answer 24

specific to Arg/Lys, proteolytic enzyme

charged side chain ends up at C terminal end of peptides

Question 25

chymotrypsin

Answer 25

cuts chain after aromatic amino acid residues (Phe, Try, Tyr)

aromatic amino acids end up at C terminal ends of peptides

Question 26

cyanogen bromide

Answer 26

cleaves proteins at internal methionine residues unlike the other proteolytic enzymes that cleave at the C terminal

Question 27

Edman method

Answer 27

used to determine amino acid sequence of peptides and proteins, removes N terminal amino acid allowing for protein with 10-40 amino acids to be determined in 30 min

Question 28

What is produced by cleaveage of a protein by reagents?

Answer 28

A mixture of peptides

Question 29

what are antibodies

Answer 29

immunoglobulins, recognize antigens, have a quaternary structure

Question 30

structure of antibodies

Answer 30

y shaped structure including heavy and light chain that are connected by disulfide bridges

Question 31

Fab vs Fc region of antibodies

Answer 31

Fab regions for antigen binding and a c terminal fragment called the Fc region

Question 32

epitope

Answer 32

specifc group or cluster of amino acids on the target molecule that an antibody recognizes

Question 33

polyclonal vs monoclonal antibodies:

Answer 33

polyclonal antibodies bind to different areas of the target molecule, while monoclonal antibodies will only bind a single specific site

Question 34

ELISA (enzyme-linked immunosorbent assay)

Answer 34

based on reactions between proteins and antibodies, used to detect presence of a ligand, has a direct and indirect method

Question 35

direct ELISA

Answer 35

primary antibody is used to bind to antigen, less intensive signals are produced, less sensitive

Question 36

indirect ELISA

Answer 36

secondary antibody conjugated with detection enzyme, primary antibody also involved, produces more intensive signals,

used to detect presence of antibody

ex. HIV test

Question 37

indirect ELISA vs sandwich Elisa

Answer 37

indirect: rate of color formation proportional to amount of antibody

sandwich: rate of color formation proportional to amount of antigen

Question 38

Western blotting

Answer 38

technique where proteins are separated using gel electrophoresis and then transferred to a nitrocellulose membrane for analysis and identification

Question 39

western blotting steps

Answer 39

uses SDS gel to transfer proteins, which are then moved to polymer sheet. A mixture of antibodies is washed and the secondary antibody is added to detect the primary antibody.

Question 40

function of flourescent markers

Answer 40

allow for visualization of proteins in the cell

Question 41

flourescence microscopy

Answer 41

allows for detection of structure/function