biochem 12

Protein separation methods

differential centrifugation, salting out

gel filtration chromatography, ion-exchange chromatography, affinity chromatography

Differential centrifugation

Separates soluble and particulate fractions of cell lysate

Salting out

Separates proteins based on solubility using high salt concentrations

Gel filtration chromatography

Separates proteins based on size and shape

Ion-exchange chromatography

Separates proteins based on net charge at a given pH

Affinity chromatography

Separates proteins based on specific ligand binding

Protein characterization

Determination of physical and chemical properties of proteins

Molecular weight determination

Can be measured using SDS-PAGE or gel filtration

Isoelectric point (pI)

pH at which a protein has no net charge

Isoelectric focusing

Technique to separate proteins based on pI in a pH gradient

UV-visible spectroscopy

Method to analyze protein absorbance properties

3D protein structure determination

Determining full structure using X-ray, NMR, cryo-electron microscopy

Polyacrylamide gel electrophoresis (PAGE)

Technique to separate proteins in a gel using an electric field

SDS-PAGE

Electrophoresis method that separates proteins based on size

• SDS gives all proteins uniformly negative charge

• small proteins move faster

Gel filtration vs SDS-PAGE similarity

Both separate proteins based on size

Gel filtration vs SDS-PAGE difference

Gel filtration uses native proteins while SDS-PAGE uses denatured proteins

Protein migration in SDS-PAGE

Proteins move toward positive electrode

Large vs small proteins in gel filtration

Large proteins elute first

Purpose of reducing conditions

Reveal subunit composition of proteins

Isoelectric focusing

Separation of proteins in a pH gradient based on pI

Proteins migrate until they reach pH where net charge is zero

Two-dimensional electrophoresis

Combines isoelectric focusing (by pI) and SDS-PAGE (by size)

Spectroscopic detection of proteins

Measurement of protein concentration using light absorbance

Aromatic amino acids

Absorb UV light in proteins

Strongest chromophores in proteins

Tryptophan and tyrosine

how to determine highest pI value?

highest molecular weight?

highest pI value: farthest to right

highest molecular weight: highest up

Lambert-Beer equation

A = ε·c·l

Relationship between absorbance and concentration of a solution

Specific activity

Enzyme activity per amount of protein (U/mg)

Measure purity of protein during purification- increases as purification occurs

Visual evaluation of purification

SDS-PAGE shows fewer bands as purity increases

Protein structure determination methods

Techniques used to determine 3D protein structure

X-ray crystallography

Determines structure from diffraction of X-rays through protein crystals

Steps in X-ray crystallography

1. purify protein

2. crystallize protein (difficult)

3. collect diffraction data

4. calculate electron density

5. fit residues into density

pros:

no size limit

well established

cons:

difficult for membrane proteins

cant see hydrogen

Advantage of X-ray crystallography

No size limit and well-established method

Limitation of X-ray crystallography

Difficult crystallization and cannot see hydrogens

Nuclear magnetic resonance (NMR)

Determines protein structure in solution using magnetic fields

Steps in NMR

Steps needed

• Purify the protein

• Dissolve the protein

• Collect NMR data

• Assign NMR signals

• Calculate the structure

Advantage of NMR

No need for crystals and can observe hydrogens and dynamics

Limitation of NMR

pros:

• Works best for small proteins

• no need to crystallize protein

cons:

difficult for insoluble proteins

Cryo-electron microscopy (Cryo-EM)

Determines protein structure using frozen samples and electron microscopy

Steps in Cryo-EM

1. purify protein

2. place in em grid and freeze

3. collect images in microscope

4. calculate 3d structure

pros:

• good for large proteins/complexes

• requires little sample and no special treatment

cons:

• new method, bad for small proteins

Advantage of Cryo-EM

Good for large proteins and complexes

Limitation of Cryo-EM

Not suitable for small proteins and equipment is expensive