Key Concepts of Amino Acids and Protein Purification Techniques

The side chains of these amino acids are most likely found in the protein interior

Ala, Cys, Phe, Ile, Leu, Met, Pro, Val, Trp, Tyr

All the amino acids with side chains that are ionizable between pH 3 and 12

Cys, Asp, Glu, His, Lys, Arg, Tyr

Acidic and basic amino acids

Asp, Glu, His, Lys Arg

Polar amino acids that are not charged

Asn, Ser, Thr, Gln

All of the polar amino acids

Asn, Ser, Thr, Gln, Asp, Glu, His, Lys Arg

All the aliphatic amino acids

Ala, Ile, Leu, Val

Amino acids that have notable effects on the polypeptide backbone conformations

Gly, Pro

All the aromatic amino acids

Phe, His, Trp, Tyr

Side chain contains three nitrogens, pKa ~12

R

Side chain amide of glutamate

Q

Oxidizes to Cross-link polypeptides

C

Absorbs UV(280 nm) strongly

W

Lacking an amide hydrogen when in a peptide

P

The only non-chiral amino acid

G

sidechain pKa is closest to physiological pH

H

Smallest amino acid with a side chain

A

Side chain pKa ~4

D

Determining the approximate number of amino acids (length is related to.....)

SDS-PAGE

Determining the molecular size of a protein's native state

Size Exclusion (gel filtration) chromatography

Accurately determining a protein's pI

Isoelectric focusing electrophoresis (IEF)

Characterizing a complex protein mixture (e.g. cell lysate) for differences in composition and amounts of individual proteins

2d electro.

Assaying binding of another protein or DNA to a purified protein of interest

native electr.

Partially purifying a basic protein from a mixture of mostly acidic proteins

ion xchange chr

Purifying a small-molecule binding protein from a complex mixture in one step

affinity chr

Enrichment of a protein in a complex mixture based on salt-dependent solubility

ammonium sulfate precipitation

Separating mitochondrial proteins from soluble cytosolic proteins

gradient ultracentrif.

Separating a lipid-binding protein from other proteins

hydrophobic interaction chr

pkas

nitro 8

carboxyl 4

asp 4

glu 4

arg 12

lys 11

his 7

cys 9

tyr 10

uv abs at 280

W

acid/base at ph 7

H

side chain contains two methylenes, an amide group

Q

hydroxyl containing side chain nearly the same shape as valine

T

the only non chiral aa

G

side chain contains one ch2 group, pka around 4

D

aro side chain with hydroxyl group

Y

isomeric with isoleucine

L

cross links polypeptides in presence of O2

C

side chain contains a primary amine, pka~11

K

can form strong covalent bonds with metal ions

H or C

first aa in a newly translated protein

M

hydrophobic side chain with nearly the same shape as threonine

V

Most hydrophobic amino acid

W

nonpolar aas

Glycine, Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan, Methionine, Proline

charged aas

aspartate, glutamate, lysine, arginine, histidine

polar uncharged aa

serine, threonine, cysteine, asparagine, glutamine

aro aas

FWY

HA amine

1/1+10^ph-pka

A cooh

10^ph-pka/1+10^ph-pka