CHM 365 Appendix

What influences protein stability?

pH, temperature, pressure, proteases

(store at low temps, use protease inhibitor cocktail, add cryoprotectants to prevent cystal formation, add pH buffers and a dash of salt

ELISA

1. immobilize first antibody on solid support

2. incubate with protein containing sample

3. add second antibody that is covalently linked to an assayable enzyme

4. wash and assay enzyme, amount of substrate → product indicates amount of protein present

Beer Lambert Law

The amount of light absorbed is directly proportional to the concentration of solute and the distance the light travels through the sample

F, Y, W absorb in UV

What separates Proteins by solubility?

Salting Out

What separates Proteins by ionic charge?

Ion Exchange Chromatography, Electrophoresis, Isoelectric Focusing

What separates Proteins by Polarity?

Hydrophobic Interaction Chromatography

What separates Proteins by size?

Gel Filtration Chromatography

SDS-PAGE

What separates Proteins by binding specificity?

Affinity Chromatography

Fractionation by Salting Out

solubility dependent

at low ion concentration→salting in: small amount of salt blocks ionic interactions between proteins

at high ion concentration→salting in: an increase in salt restults in salting out (precipitation) that reduces bulk H20 and HE

for salting out pH=pI (least soluble)

Ion Exchange Chromatography

matrix can be anion exchanger (+ charge→DEAE) or cation exchanger (- charge→carboxymethyl)

proteins interact via charge and as ionic strength increases, more proteins elute

Gel Filtration Chromatography

separates by size and shape

contains beads with pores, larger molecules will elute first

can be used to determine the MW of a protein

Hydrophoic Interaction Chromatography

stationary phase contains acetyl or phenyl groups

HE increases as the ionic strength increases

load protein with a lot of salt, resulting in salting out

Affinity Chromatography

separates based on ligand binding or immunoaffinity

add His Tag that will chelate Ni2+ and bind it to the stationary phase

get protein off by adding free Ni2+ or His

Gel Electrophoresis

Separates by size/shape/and charge at pH 9

use polyacrylamide gel and load protein at cathode (-), which will migrate towards anode (+)

SDS-PAGE

separates by mass

deantures proteins and molecules with lower MW move more rapidly

molecules with disulfide bonds are more compact, resulting in faster migration (break using BME)

2-D Gel Electrophoresis

combination of IEF (Isoelectric Focusing) and SDS PAGE→ separates MW and pI

protein settles when pI=pH, then run SDS PAGE perpendicular

Why do we do protein sequencing?

to understand structure/function relationships, sequence comparison, diagnostic tests and therapies

What can be used to reduce disulfide bonds to separate protein strands?

BME, DTT, iodoacetate (blocks disulfide bond formation)

What are chemical and enzymatic methods to break a polypeptide?

Chemical-cyanogen bromide

Enzymatic- endopeptidase

Trypsin

cleaves C term of pos charged residues (Arg, Lys) as long as proline is not next to it

Chymotrypsin

cleaves C term of bulky hydrophobic residues (Phe, Trp, Try) as long as proline is not next to it

Elastase

cleaves C term of small neutral residues (Ala, Gly, Ser, Val) as long as proline is not next to it

Electrospray Ionization (ESI) Mass Spec

proteins are intact and are atomized under acidic condition and are analyzed to get mass to charge ratio

Tandem Mass Spec

goes through MS1 and is filtered, and then strands are fragmented by collision cell (He) and undergo MS2