Protein activity and monitoring purification

What is protein activity

A measure of what a protein does; for an enzyme, it is commonly assessed by measuring the rate of a catalysed reaction.

What is an enzyme assay

A quantitative measurement of an enzyme’s reaction rate under specified conditions.

Why should enzyme activity be measured under controlled conditions

Temperature, pH, substrate concentration, and other conditions affect reaction rate and help determine whether differences reflect enzyme amount or changes in conditions.

What is an enzyme unit

In this slideshow, the amount of enzyme that transforms (1.0\ \mu\text{mol}) of substrate per minute at (25^\circ\text{C}).

What is total enzyme activity

The total measured activity of the enzyme in a sample or fraction.

What is specific activity

Enzyme activity per unit mass of total protein, commonly expressed as units per milligram of protein.

How is specific activity calculated

Specific activity = total enzyme activity ÷ total protein mass.

Why should specific activity increase during enzyme purification

Contaminating proteins are removed while the enzyme of interest is retained, increasing the proportion of the total protein represented by the enzyme.

What does an increase in specific activity indicate

The preparation is becoming enriched in the active enzyme.

When is a protein generally considered pure according to the slideshow

When additional purification no longer increases its specific activity and it produces a single band on SDS-PAGE.

Why is a protein assay necessary during purification

It measures protein concentration or amount so that purification fractions and specific activity can be evaluated.

What is gel electrophoresis

A technique that separates molecules as they move through a gel in an electric field.

What protein properties can gel electrophoresis exploit

Depending on the method, protein charge, molecular mass, and isoelectric point.

What is PAGE

Polyacrylamide gel electrophoresis, a method used to separate and characterize proteins in a polyacrylamide gel.

What is SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis, a method commonly used to estimate protein molecular mass and examine polypeptide subunits.

What does SDS do to proteins

It unfolds proteins and gives them a broadly similar negative charge-to-mass ratio, allowing separation mainly according to molecular mass.

How does SDS alter protein shape

It disrupts much of the protein’s native structure, causing the polypeptide to become unfolded.

How does SDS alter protein net charge

It binds along the polypeptide and gives it a strong negative charge that largely overwhelms its original charge.

Why does SDS-PAGE separate proteins mainly by molecular mass

SDS reduces differences in native shape and charge, so smaller polypeptides generally migrate through the gel faster than larger ones.

How is molecular mass estimated using SDS-PAGE

Compare the migration of a protein band with molecular-mass standards, or a protein ladder, run on the same gel.

What is a molecular-mass ladder

A mixture of proteins of known molecular masses used to estimate the molecular masses of unknown proteins.

What does a single band on an SDS-PAGE gel suggest

The sample may contain one dominant polypeptide species, although a single band alone does not prove complete purity.

What does the presence of multiple SDS-PAGE bands suggest

The sample may contain multiple polypeptides, contaminants, or different processed or degraded forms of a protein.

How can proteins be visualized after electrophoresis

By staining the gel with protein-binding dyes or other reagents, or by using antibody-based detection such as Western blotting.

What is Coomassie staining

A protein-staining method used to visualize protein bands in a gel.

What is silver staining

A gel-staining method that uses silver-based chemistry and is generally more sensitive than Coomassie staining, according to the slideshow.

What is Western blotting

A method in which proteins are transferred from a gel to a membrane and detected using antibodies that recognize a specific protein.

What is the main advantage of Western blotting over general protein staining

It can detect a particular target protein among many proteins in a sample.

What is isoelectric focusing (IEF)

A technique that separates proteins according to their isoelectric points using a pH gradient and an electric field.

How does a protein behave during isoelectric focusing

It migrates through the pH gradient until it reaches the pH at which its net charge is approximately zero.

What is the main property measured by IEF

The protein’s isoelectric point (pI).

What is two-dimensional electrophoresis (2D electrophoresis)

A technique that separates proteins in two dimensions, typically first by pI using IEF and then by molecular mass using SDS-PAGE.

What information does 2D electrophoresis provide that SDS-PAGE alone does not

It separates proteins by both pI and molecular mass, allowing proteins with similar molecular masses but different pIs to be distinguished.

What information does 2D electrophoresis provide that IEF alone does not

It adds molecular-mass separation to separation by pI.

How do SDS-PAGE and IEF differ

SDS-PAGE primarily separates unfolded polypeptides by molecular mass, whereas IEF separates proteins by their pI.

How are SDS-PAGE and IEF similar

Both use an electric field and a gel-based system to separate proteins, but they exploit different protein properties.

What can a 2D electrophoresis gel reveal about a complex protein mixture

It can resolve many different protein species into separate spots according to their pI and molecular mass.

How can SDS-PAGE reveal protein subunit composition

Comparing samples run with and without a reducing agent can help reveal whether subunits are connected by disulfide bonds.

What does a reducing agent such as β-mercaptoethanol (BME) do in SDS-PAGE sample preparation

It reduces disulfide bonds, allowing polypeptide chains linked by those bonds to separate.

If a protein produces different band patterns with and without BME, what can you infer

Disulfide bonds may be connecting polypeptide chains or maintaining covalent links between parts of the protein.