Isolating & analyzing proteins

Question 1

what is subcellular fractionation?

Answer 1

process of isolating cellular components whilst keeping their functions intact

Question 2

what is the most distinctive property of a protein that can be used to separate it?

Answer 2

function

Question 3

What are the two most common ways to separate proteins based on size and charge?

Answer 3

size exclusion chroma and ion exchange chroma

Question 4

what is size exclusion chroma?

Answer 4

column packed with porous polymer beads, protein run through using solvent and separated out based on size. Large come out first, smaller partition in out more

Question 5

what is ion exchange chroma?

Answer 5

uses a charge at a particular pH, beads are either positively or negatively charged
things that don’t stick to column elute first
add salt to make positive charged proteins be dislodged

Question 6

What is affinity chroma?

Answer 6

if the function of the protein is known and what helps it perform that function an affinity tag can be made which is then put on beads. The unwanted proteins are then washed off because the substrate etc… is bound to the beads. The protein of interest is washed off by using a ligand solution
best method

Question 7

Can you add an affinity tag to a recombinant protein for expression and purification?

Answer 7

YES

Question 8

what happens in electrophoresis? (vague)

Answer 8

charged proteins are separated based on their different mobilities in an electric field

Question 9

what does SDS-PAGE stand for?

Answer 9

SDS polyacrylamide gel electrophoresis

Question 10

what does SDS-PAGE separate based on?

Answer 10

negative charged sds-protein micelles based on their size and thus their molecular weight

Question 11

in SDS-PAGE what size proteins migrate fastest toward the + electrode?

Answer 11

small!!

Question 12

why is a reducing agent added? (sds page)

Answer 12

to cleave S-S bonds and resolve individual polypeptide chains

Question 13

What is a 2-D page?

Answer 13

separates by both pH and size (1 d ph, 2 d MW)

can reveal hundreds of proteins and disease related difference in protein levels

Question 14

Can proteins be sequenced using mass spectrometry?

Answer 14

YES. how fast they fly depends on their charge and inversely on their mass

Question 15

what is trypsin used for in mass spectrometry?

Answer 15

to cleave the peptide bonds at lys and arg

Question 16

what is peptide mass fingerprinting?

Answer 16

use trypsin to cleave and then get a mass spec which you use to compare to the theoretical masses for all trypsin released proteins in a data base

Question 17

what is tandem MS-MS?

Answer 17

after trypsin digestion mass spectrometer gives peptide masses which are then further fragmented at their peptide bonds. Once fragmented more a coupled spec is created giving the fragmented masses. The mass difference between fragments can be used to determine a partial AA sequence which can then be used gene identification or gene cloning

Question 18

What tool determines the 3D arrangement of atoms in a protein starting of with a crystal?

Answer 18

X-ray crystallography
hit crystal with at varying angles with x rays to get a diffraction pattern. You can then work out the electron density map which provides details on conformation binding partners and enzyme function

Question 19

What tool has contributed the most structures?

Answer 19

Xray crys.

Question 20

What is a problem with x ray crystallography?

Answer 20

get a somewhat static view of the proteins structure because it is in its crystal form which is not representative of how it behaves in aqueous solution

Question 21

what does NMR stand for?

Answer 21

nuclear magnetic resonance

Question 22

what is a benefit of NMR?

Answer 22

don’t need crystal form of protein–works in solution

but it has to be pure and all that is present in that solution

Question 23

what nuclei spins are measured in NMR?

Answer 23

1H, 13C, 15N

Question 24

what can the electromagnetic resonance of frequency of nuclei used for?

Answer 24

to provide information on the neighbouring atoms, through bonds or space

Question 25

What does NMR generate?

Answer 25

the possible conformations of the protein that fit the data–not just one answer
tells you what parts of the protein are flexible which has to do with function
only found 10% of structures this way

Question 26

Why do we want to solve the 3-D structure of proteins?

Answer 26

tells us about function
tells us how mutations may affect function
development of drugs–create molecules that inhibit a particular enzyme in vitro