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GSBS Biochem > Proteins 3 > Flashcards

Proteins 3 Flashcards

1
Q

types of chromatography

A

size exclusion
hydrophobic interaction
ion exchange
affinity
reverse phase

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2
Q

how does size exclusion chromatography work

A

Pores in beads exclude larger molecules but smaller ones are
retarded - i.e. the volume in the column is larger for smaller
molecules

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3
Q

why monitor at 280nm?

A

thats where aromatic amino acids such as tryptophan and tyrosine absorb

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4
Q

Things that can degrade resolution (i.e. broaden peaks);

A

high solute concentrations, high flow rate, large sample volume, column needs to be repacked.

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5
Q

size exclusion chromatography issues

A

charge and shape issues

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6
Q

main detergents used

A

OG - octyl glucoside
DDM
Cymal - 5
Triton X 100

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7
Q

main thing to think about when using detergents

A

CMC

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8
Q

size exclusion pros

A

1) Can use high salt
2) Can use detergent
(below CMC)
3) Sizing often works to
some degree
4) Probably the only way
to separate oligomers
of the same protein (ehhh)

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9
Q

size exclusion cons

A

1) Dilutes the sample
2) Relatively slow
3) Slow turnaround
4) Resolution-variable
5) Sensitive to [Protein]
6) Low loading volume

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10
Q

ion exchange chromatography

A

a charged column grabs other charged proteins

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11
Q

two positively charged column types

A

Mono Q (FPLC, high resolution, comes as pre-packed columns)
DEAE, diethylaminoethyl cellulose

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12
Q

two negatively charged columns

A

Mono S (FPLC, high resolution, comes as pre-packed columns)
CMC, carboxymethyl cellulose

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13
Q

two kinds of buffers for ion exchange chromatography

A

positive and negative

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14
Q

when making phosphate buffer, do you need to mix all forms of phosphate to get to a certain pH?

A

NOOOOO

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15
Q

ion exchange pros

A

1) Can concentrate sample
2) Can use with detergents
(non-ionic)
3) Can resolve very well
4) Relatively fast (e.g. <1 hour
per run start to finish with
FPLC)

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16
Q

ion exchange cons

A

1) Does not work with
proteins that require
salt for solubility.

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17
Q

hydrophobic interactions are strengthened in

A

high salt

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18
Q

in hydrophobic interaction, elution occurs as

A

salt is decreased

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19
Q

problem with hydrophobic interaction

A

protein may never come off

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20
Q

natural affinity chromatography means

A

taking proteins from the natural source and purifying with small molecular weight ligands such as GTP, ATP, NAD(P), etc.

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21
Q

pros of natural affinity chromatography

A

1) Can concentrate the sample
2) Can have high yield - depending
upon contaminants
3) Can be a crude way to look at
protein binding - e.g. does a
mutant block binding.

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22
Q

cons of natural affinity chromatography

A

1) Can be a common ligand that
binds lots of stuff.
2) Linker to beads is critical.
Some linkages will prevent
binding to protein.
3) Elution can be costly. E.g.
maybe ATP is the only way to
elute ($$$$)
4) Usually not one step
purification.

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23
Q

examples of expression affinity tags

A

his tag
maltose binding protein
glutathione S transferase
antigens

24
Q

how to remove a tag

A

can use Tobacco etch virus protease (TEV) to cut at a specific site

25
Q

how is TEV normally used

A

1) Express protein of interest.
2) Purify with the tag (e.g. His tag and nickel affinity columns)
3) Add rTEV (usually overnight at 4°C)
4) Pass over nickel column to get rid of the rTEV protease.

26
Q

affinity tags pros

A

1) Can concentrate the
sample
2) Can have high yield
3) Can yield high purity

27
Q

affinity tags cons

A

1) Not all proteins can
accommodate the tags
2) Go for cheap elution
tags…

28
Q

equilibrium ultracentrifugation separates via

A

density

29
Q

non-equilibrium untracentrifugation separates via

A

density and size

30
Q

examples of equilibrium ultracentrifugation medium

A

CsCl, K tartrate

31
Q

examples of non-equilibrium ultracentrifugation medium

A

sucrose, glycerol, Ficoll-Paque

32
Q

equilibrium ultracentrifugation pros

A

1) Can concentrate sample
2) Can yield high purity

33
Q

equilibrium ultracentrifugation cons

A

1) CsCl can be harsh
2) Takes >24hrs

34
Q

non-equilibrium ultracentrifugation pros

A

1) Gentler to sample
2) Can remove a great deal of
debris
3) Faster (1-2 hours)

35
Q

non-equilibrium ultracentrifugation cons

A

1) Will not concentrate
sample as much.
2) Not as sensitive as
density purification.

36
Q

the best way to look ar oligomeric state is with

A

analytical centrifugation

37
Q

what is agarose

A

highly purified polysaccharide derived from agar, long sugar polymers held together by hydrogen and hydrophobic bonds

38
Q

what is acrylamide

A

polyacrylamide gel structure held together by covalent cross-links

39
Q

why should you not trust MW on SDS gels

A

Some proteins are VERY tightly packed and hard to reduce
even if boiled with SDS and BME
Highly charged proteins are hard to ‘neutralize’ with SDS.
May still run too fast or too slow.
Many teach to plot Rf vs Log(MW). Meh. Everything is a
straight line in a Log plot.

40
Q

2D electrophoresis

A

First dimension, you run an IEF. Separate native proteins via charge. You
then lay that IEF strip onto an SDS-PAGE to separate denatured proteins
via size. The spots can be isolated, digested, and identified via MS

41
Q

western blot chromogenic reactions

A

are very useful
since they do not require any special equipment for development. However,
they are not very sensitive and can be more difficult to quantify.
Alkaline phosphatase: NBT, BCIP,
Fast Red AS-MX.
Horseradish peroxidase: 4-CN, DAB, and TMB. I tend to favor these since HRP is more stable and these substrates are relatively inexpensive.

42
Q

western blot chemiluminescence reactions

A

Reaction can be more sensitive and more quantitative. Not as easy or as fast as chromogenic and requires x-ray film, image
plate, or CCD technology.

43
Q

three types of chemical precipitation

A

acetone
ammonium sulfate
polyethylene glycol

44
Q

why not use acetone for chemical precipitation

A

very harsh, lots of waste

45
Q

problems with polyethylene glycol

A

Polyethylene glycol;
Hard to get rid of - best used on viruses

46
Q

ammonium sulfate chemical precipitation

A

Can either stabilize or destabilize protein
Can effectively concentrate from large volumes
Can use sequential cuts to remove contaminants
Easy to remove via dialysis
However, may lose protein if too dilute.

47
Q

centrifigal concentrators

A

Wide range of volumes and MW
cutoffs
Not effective with very large
volumes

48
Q

tangential flow

A

Like centrifuge
concentrators - pressure
pushes small MW
compounds through.
Here, the tangental flow of
material keeps the filter
from clogging.
Expensive but the only
way to concentrate large
volumes

49
Q

what buffer could you use to refold inclusion bodies

A

glutathione redox buffer

50
Q

refolding example steps

A

expression
isolation and denaturation of inclusion bodies
protein refolding via redox buffer
protein purification
his tag removal by TEV
size exclusion chromatography

51
Q

how to detect proteins without enzymatic activity

A

western blots (if antibody is available)
fluorescent protein

52
Q

what is the first purification method you would try with a very basic protein

A

E. coli expression

53
Q

what is the pI of a protein and how do you measure it

A

pI of the protein is the pH at which the net charge of the protein is zero. it is measured via isoelectric focusing (IEF)

54
Q

why would a protein band shift in apparent molecular weight in SDS page with or without a reducing agent

A

dimers
intermolecular or intramolecular disulfides

55
Q

how would phosphorylation affect SDS page

A

phosphorylation would add an extra negative charge, would change movement

56
Q

how would glycosylation have an effect on SDS page

A

smeared band because of natural heterogeneity in the sugars.

GSBS Biochem (13 decks)

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