M7 protein separation and purification

Why isolate and purify a protein?

To analyze its biochemical properties, activities, structure, and interactions.

What is the general principle of protein purification?

Exploit unique properties of proteins (size, charge, solubility, binding) while preserving function.

What is the first step in protein purification?

Lyse cells to release proteins (e.g., with sonicator or French press).

After lysis, how is crude extract prepared?

Centrifugation removes cell debris, leaving soluble proteins in the supernatant.

What is ammonium sulfate precipitation?

A method that alters solubility using salt concentration to selectively precipitate proteins.

What is “salting in” vs. “salting out”?

At low salt: solubility increases (“salting in”);

at high salt: solubility decreases and proteins precipitate (“salting out”).

Why is ammonium sulfate often used in precipitation?

It is highly soluble and strongly affects protein solubility.

What is column chromatography?

A separation method where proteins move through a resin-filled column and separate based on properties.

What are three main types of chromatography?

Size-exclusion, ion exchange, and affinity chromatography.

How does size-exclusion chromatography work?

Small proteins enter pores and travel slowly;

large proteins bypass pores and elute faster.

find large first

How does ion exchange chromatography work?

Proteins bind to charged resins depending on their net charge;

elution is controlled by salt or pH.

What factors affect ion exchange binding?

Net protein charge

pH of solution

ionic strength (salt concentration).

How does affinity chromatography work?

Proteins bind specifically to ligands (e.g., antibodies, cofactors, inhibitors) attached to resin.

What is PAGE used for?

separates proteins by size and charge

What is SDS-PAGE?

denatures protein with SDS

gives uniform (-)

separation by molecular mass

What is isoelectric focusing (IEF)?

Separation of proteins in a pH gradient until they reach their isoelectric point (pI).

What is two-dimensional gel electrophoresis?

Combines isoelectric focusing (separation by pI) and SDS-PAGE (separation by size) for high-resolution analysis.

mass and pI

solubility

disrupt cell membranes and create crude extract

precipitation with ammonium sulfate (salting out)

size/shape

size-exclusion chromatography

isoelectric point (charge)

ion exchange chromatography

binding to small molecules

affinity chromatography

sonicator

sound waves disrupt cell membranes

french press

pressure disrupt cell membranes

soluble lysate

decant off supernatant with soluble protein

cation exchanger (- resin) binds

cations (+) proteins

anion exchanger (+ resin) binds

anions (-) proteins

in ion exchange chromatography, proteins with ___ charges elute first

smaller

the greater the net charge on a molecule, the ___ the protein binds

better

steps for 2 dimensional gel electrophoresis

put charged proteins in gel with pH gradient

stop at pI values

denature, SDS, stop on MW

if you were bringing down hemoglobin in a salt concentration what molecule would you use to do it?

ammonium sulfate

in ion exchange chromatography, molecules with the largest (-) charge are going to come out first if you have a resin that has a

(-) charge