Protein Purification and Analysis Techniques

Acidic Amino Acid Net Charge at $\text{pH} = 7$

A net negative charge of $-1$ at $\text{pH} = 7$.

Basic Amino Acid Net Charge at $\text{pH} = 7$

A net positive charge of $+1$ at $\text{pH} = 7$.

Protein Net Charge at $\text{pH} = \text{pI}$

No net charge ($0$).

Protein Net Charge when $\text{pH} > \text{pI}$

A net negative charge.

Protein Net Charge when $\text{pH} < \text{pI}$

A net positive charge.

Ion- exchange chromatography

Setting the buffer $\text{pH}$ below the protein's $\text{pI}$ ($\text{pH} < \text{pI}$), which gives the protein a net positive charge to bind to the negatively charged cation exchanger resin.

Elution by Increasing Salt Concentration

A method to elute proteins from ion-exchange resins where salt ions increase ionic strength and compete with the protein for binding sites on the resin.

Elution by Altering Buffer $\text{pH}$

A method to elute proteins from ion-exchange resins by changing the buffer $\text{pH}$ toward the protein's $\text{pI}$, weakening electrostatic binding to the resin.

Cellulosic Ion-Exchanger Operating Conditions

A $\text{pH}$ range of $5$ to $9$ and low ionic strength, under which protein affinity for the resin decreases as ionic strength increases.

First Dimension of 2D Gel Electrophoresis

Isoelectric Focusing (IEF), which separates proteins by their isoelectric point ($\text{pI}$).

Second Dimension of 2D Gel Electrophoresis

SDS-PAGE, which separates proteins by molecular weight or size ($M_r$).

Early Low-Resolution Purification Steps

Initial purification techniques consisting of heat treatment and ammonium sulfate fractionation.

Enzyme Unit

The amount of enzyme that catalyzes the conversion of $1\,\mu\text{mol}$ of substrate per minute under specified conditions.

Specific Activity

The units of enzyme activity per milligram of total protein.

Enzyme Yield

The percentage of initial total enzyme activity retained at a specific stage of purification.

Completion Criteria for Purification

Reaching a state where a single spot is produced on a 2D gel electrophoresis.

Gel Type Selection for Size Exclusion

Choosing a pore size where the target protein is retarded by the gel matrix but not completely excluded (falling within the gel's fractionation range).

Purpose of Monitoring $\text{O.D.}_{280}$ ($A_{280}$)

Measuring UV light absorption at $280\,\text{nm}$ by aromatic amino acids to allow rapid, non-destructive estimation of protein concentration in fractions.

Ion-Exchange Separation Basis

Net charge.

Gel Filtration Separation Basis

Native size / molecular weight.

SDS-PAGE Separation Basis

Subunit molecular weight.

Original sample identity and reasoning

Identity: homodimernof two 40 kD subunits held together by a disulfide bond

Gel filtration

Shows native 80 kD diners by dissociated 40 kD monomers

SDS page without b-m (80+40)

Non covalent bonds break, intact disulfide bonds preserve the 80 kD covalently linked dimer

SDS page with b-m (single 40 kD)

b-me reduces disulfide bonds converting all 80 kD complexes into identical 40 kD subunits