Protein Analysis Techniques (Concepts)

What property does SDSPAGE separate proteins by?

Molecular weight (size)

What are the two main functions of SDS in SDSPAGE?

Denatures proteins and gives them a uniform negative charge

Why does SDSPAGE separate proteins by size rather than charge?

SDS gives all proteins a similar charge to mass ratio

In SDSPAGE, which proteins migrate farthest through the gel?

Smallest proteins

A protein migrates farther on an SDSPAGE gel. What can be concluded?

It has a lower molecular weight

What property does isoelectric focusing separate proteins by?

Isoelectric point (pI)

What is the isoelectric point (pI)?

The pH at which a molecule has no net charge

When does a protein stop moving during isoelectric focusing?

When it reaches a region where pH equals pI

A protein with pI = 9 is placed in pH 7. What is its net charge?

Positive

A protein with pI = 4 is placed in pH 7. What is its net charge?

Negative

If pH is less than pI, what is the protein's charge?

Positive

If pH is greater than pI, what is the protein's charge?

Negative

What property does Ionexchange chromatography separate molecules by?

Net charge

How does Ionexchange chromatography work?

Molecules bind to oppositely charged groups on the stationary phase

Why can increasing salt concentration elute proteins from an Ionexchange column?

Salt ions compete for ionic interactions

A positively charged protein will bind to what type of Ionexchange resin?

Negatively charged resin

A negatively charged protein will bind to what type of Ionexchange resin?

Positively charged resin

What property does Sizeexclusion chromatography separate molecules by?

Size

Which molecules elute first during Sizeexclusion chromatography?

Large molecules

Why do large molecules elute first in Sizeexclusion chromatography?

They enter fewer pores in the beads

Which molecules spend more time inside the pores of the stationary phase in Sizeexclusion chromatography?

Small molecules

What property does Affinity chromatography separate molecules by?

Specific binding interactions

A protein specifically binds ATP. Which purification technique is best?

Affinity chromatography

How are proteins typically eluted from an Affinity column?

By adding free ligand or changing conditions to disrupt binding

What is the stationary phase in chromatography?

The phase that remains fixed

What is the mobile phase in chromatography?

The phase that moves through the stationary phase

What determines separation in chromatography?

Different interactions with the stationary and mobile phases

What property is primarily exploited in paper chromatography?

Differences in polarity and solubility

What property is primarily exploited in TLC?

Differences in polarity and solubility

In TLC, what does a higher Rf value indicate?

Greater affinity for the mobile phase and lower affinity for the stationary phase

What is the equation for Rf?

Distance traveled by compound divided by distance traveled by solvent front

What does electrophoresis separate molecules by?

Movement in an electric field based on charge and size

During electrophoresis, negatively charged molecules migrate toward which electrode?

Positive electrode (anode)

During electrophoresis, positively charged molecules migrate toward which electrode?

Negative electrode (cathode)

What is the purpose of a Western blot?

Detect a specific protein

What molecule is detected in a Western blot?

Protein

What molecule is detected in a Southern blot?

DNA

What molecule is detected in a Northern blot?

RNA

What is transferred to a membrane during Western blotting?

Proteins

What is used to identify a protein in a Western blot?

Specific antibodies

What is the typical order of Western blotting?

SDSPAGE then transfer to membrane then antibody detection

What is ultracentrifugation?

A technique that separates particles based on sedimentation behavior

What property determines sedimentation during ultracentrifugation?

Size, shape, and density

Which particles generally sediment faster during ultracentrifugation?

Larger and denser particles

What does mass spectrometry measure?

Mass to charge ratio (m/z)

Why must molecules be ionized before mass spectrometry?

The instrument measures mass to charge ratio

What is one major use of mass spectrometry in biochemistry?

Determining molecular mass of proteins and peptides

Which technique gives the most accurate molecular weight measurement?

Mass spectrometry

Which technique is best for determining protein purity and approximate size?

SDSPAGE

Which technique is best for determining exact molecular weight?

Mass spectrometry

Which technique is best for separating proteins by pI?

Isoelectric focusing

Which technique is best for separating proteins by charge?

Ionexchange chromatography

Which technique is best for separating proteins by size?

Sizeexclusion chromatography or SDSPAGE

Which technique is best for isolating a protein with a known binding partner?

Affinity chromatography

What does SDSPAGE tell you about a protein?

Its approximate molecular weight and purity

What does isoelectric focusing tell you about a protein?

Its isoelectric point (pI)

What does Western blotting tell you about a protein?

Whether a specific protein is present

What does mass spectrometry tell you about a protein?

Its exact molecular mass

MCAT Shortcut: SDSPAGE separates by what?

Molecular weight

MCAT Shortcut: Isoelectric focusing separates by what?

pI

MCAT Shortcut: Ionexchange chromatography separates by what?

Charge

MCAT Shortcut: Sizeexclusion chromatography separates by what?

Size

MCAT Shortcut: Affinity chromatography separates by what?

Specific binding interactions

MCAT Shortcut: Western blot detects what?

Protein

MCAT Shortcut: Southern blot detects what?

DNA

MCAT Shortcut: Northern blot detects what?

RNA