Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BioChem 1 > Protein Purification > Flashcards

Protein Purification Flashcards

1
Q

How do you lyse animal tissue cells?

A

Osmotic Lysis: put cell in hypotonic solution- osmotic pressure forces water into cell causing them to swell and rupture

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is sonication?

A

Using high frequencies of sound breaks open cell walls

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is French pressing?

A

Using high pressure to force cells through pinhole opening, sheer causes cells to lyse open

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is a lysozyme?

A

enzyme that breaks down bacteria’s cell walls

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What do you do after lysing the cell?

A

Centrifuge down cell debris and protect the protein of interest

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is added when centrifuging the cell and why?

A

Protease Inhibitors: serine and cysteine proteases degrade protein
EDTA: complex all metals
Buffer pH: keep enzyme from oxidation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is the simplest and most accurate assay?

A

Ideally protein will catalyze a reaction where product or reactants can be followed spectrophotometrically

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What happens if product or reactants are not directly observable?

A

product coupled to a second enzyme to produce an observable product

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What happens if a coupled assay can’t be developed?

A

Radio labeled substrates can be used

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are the steps of a radio labeled substrate process?

A
  1. immobilize first antibody on solid support
  2. incubate with protein containing sample
  3. add secondary antibody that is covalently linked to assemble enzyme
  4. wash and assay the enzyme
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is a salt fractionation?

A

solubility of proteins are sensitive to the concentration of dissolved salts

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is salting in?

A

at low salt concentrations proteins are attracted to each other’s ionic charges and aggregate. Solubility of protein increases as salt concentration is raised.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is salting out?

A

Opposite at high concentrations. solubility of proteins decrease as salt concentration gets higher

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Typically, how many salt concentrations are done?

A

2: one well below where protein of interest precipitates and one salt cut where all protein of interest is precipitated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is column chromotagraphy?

A

Most powerful method for fractionating proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What does column chromatography take advantage of to fractionate proteins?

A

charge, size, binding affinity, polarity

17
Q

What is the stationary phase in column chromatography?

A

porous solid material held in a column

18
Q

What is the mobile phase in column chromatography?

A

buffer solution which prelocates through the column

19
Q

What are the different types of chromatography?

A

Size Exclusion Chromatography (Gel Filtration)
Ion-Exchange Chromatography
Affinity Chromatography

20
Q

What is size exclusion chromatography?

A

separates proteins based on size, column made of tiny beads of cross linked polymers, larger proteins cannot fit into most pores and migrate faster
- Larger proteins: more direct route (faster)
- Smaller proteins: fit through the pores; come out last (slower)

21
Q

What is ion-exchange chromatography?

A

exploits difference in sign and magnitude of net charges at given pH, affinity for each protein affected by pH, optimize separation by gradually changing pH or salt concentration

22
Q

What are cation exchangers?

A

column matrixes with covalently bound anionic groups

23
Q

What are anion exchangers?

A

column matrixes with covalently bound cationic groups

24
Q

What is affinity chromatography?

A

exploits proteins based on noncovalently binding to specific molecules

25
Q

What is a ligand?

A

something that binds to protein of interest

26
Q

How do you recover the protein in an affinity chromatography?

A

recovered by adding free ligand; another substrate, changing salt concentration, or changing pH to release protein

27
Q

What is dialysis?

A

separates proteins based on size through semipermeable membranes

28
Q

What is ectrophoresis?

A

analytical tool to determine the number of proteins present and estimate the purity of the proteins. Also determine molecular weight and pI

29
Q

What is SDS-page?

A

Detergent that denatures proteins, binds through hydrophobic interactions (1 SDS per 2 amino acids), changes protein shapes into rods and separates based on molecular weight

30
Q

What is isoelectric focusing?

A

determine pI of protein

31
Q

What is dimensional electrophoresis?

A

separated by isoelectric and then separated by molecular weight

BioChem 1 (4 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions