Biochem 14.1 Protein Electrophoresis Concepts

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Questions and Answers

What is the unit used to measure the size of double-stranded DNA?

Base pairs (bp) and kilobase pairs (kbp) (correct)

Nanometers (nm)

Angstroms (Å)

Micrometers (µm)

Why is native PAGE generally more complicated for proteins than for DNA?

Proteins have varied charges due to their isoelectric points. (correct)

Proteins have a uniform size and charge.

Proteins migrate independently of their molecular weight.

Electrophoresis of proteins occurs at varying pH levels.

At what pH does native PAGE typically occur to preserve protein structure?

pH 7-7.4 (correct)

pH 4-5

pH 6-7

pH 8-9

What happens to positively charged proteins in relation to the electrodes during native PAGE?

They migrate toward the cathode. (B) Signup and view all the answers

Which of the following factors affects how proteins migrate in native PAGE?

Charge, mass, and shape (compactness) (B) Signup and view all the answers

If two proteins have the same size but different charges, which protein will likely migrate faster?

The protein with the higher charge (B) Signup and view all the answers

What characteristic of proteins can cause them to migrate differently despite having the same charge?

Their compactness or folding (D) Signup and view all the answers

What is the significance of keeping proteins intact during native PAGE?

To preserve their quaternary structure (B) Signup and view all the answers

What does a mobility shift in native gels indicate?

Interaction with a ligand (B) Signup and view all the answers

What is the primary component of the denaturing agent SDS?

Sodium ion and a negatively charged sulfate group (D) Signup and view all the answers

How does SDS influence the structure of proteins?

Disrupts hydrophobic interactions (A) Signup and view all the answers

Which technique is commonly used to separate proteins during denaturing electrophoresis?

SDS-PAGE (A) Signup and view all the answers

What aspect of protein structure does SDS not affect?

Disulfide bonds (A) Signup and view all the answers

What is the result of proteins being separated by SDS-PAGE?

Separation based only on size (B) Signup and view all the answers

In what condition can proteins separated by SDS-PAGE be used to assess activity?

Only if allowed to renature (C) Signup and view all the answers

What kind of agent is sodium dodecyl sulfate (SDS) classified as?

Denaturing agent (D) Signup and view all the answers

What effect does increasing the percentage of polyacrylamide have on the gel's pore size?

It decreases the pore size. (C) Signup and view all the answers

What type of molecules are primarily separated using agarose gels?

Large proteins, DNA, and RNA (B) Signup and view all the answers

In the context of electrophoresis, where do negatively charged particles migrate?

Toward the cathode. (B) Signup and view all the answers

What is the main purpose of adjusting the pH of the buffer in gel electrophoresis?

To change the charge of the molecules. (D) Signup and view all the answers

Why are polyacrylamide gels preferred for protein separation?

They have smaller pores suitable for small proteins. (A) Signup and view all the answers

What happens when the current is applied in an electrolytic cell during electrophoresis?

Negatively charged particles accumulate at the anode. (D) Signup and view all the answers

Which of the following correctly describes the migration of molecules in gel electrophoresis?

The smallest and most highly charged molecules migrate the farthest. (B) Signup and view all the answers

What is the role of the cathode in an electrolytic cell used for electrophoresis?

It attracts anions for reduction reactions. (B) Signup and view all the answers

What is the purpose of isoelectric focusing in protein separation?

To separate proteins based on their isoelectric point (C) Signup and view all the answers

During isoelectric focusing, what happens to proteins when they are in a medium with a pH lower than their isoelectric point?

They gain protons and become positively charged (A) Signup and view all the answers

What occurs at the point in the gel where the pH equals the isoelectric point of a protein?

The protein has zero net charge and stops migrating (C) Signup and view all the answers

Which end of the isoelectric focusing gel has a lower pH?

The cathode end (B) Signup and view all the answers

Why is isoelectric focusing typically done without denaturing agents like SDS?

To allow proteins to maintain their native structures (B) Signup and view all the answers

In isoelectric focusing, how do proteins from multiple loading positions migrate in terms of charge?

Proteins migrate based on their charge, adjusting to the pH of the gel (B) Signup and view all the answers

What is the role of the electric field in isoelectric focusing?

To facilitate the migration of charged proteins (B) Signup and view all the answers

What effect does increasing the pH of the gel have on positively charged proteins during isoelectric focusing?

They become negatively charged as they lose protons (A) Signup and view all the answers

What is the primary function of Coomassie stain in electrophoresis?

To stain proteins and make them visible (A) Signup and view all the answers

When using a gel for DNA analysis, which dye is commonly used to enhance fluorescence?

Ethidium bromide (D) Signup and view all the answers

What does a thicker and darker band in a Coomassie-stained gel indicate?

High concentration of proteins (A) Signup and view all the answers

In electrophoresis, where are samples typically loaded when dealing with negatively charged molecules?

Near the cathode (B) Signup and view all the answers

What is the purpose of including a 'ladder' lane in a gel?

To estimate the sizes of other samples (B) Signup and view all the answers

What phenomenon occurs when the electric current in an electrophoresis setup is turned off?

Molecules stop migrating (A) Signup and view all the answers

Which characteristic factors influence the migration of molecules in electrophoresis?

Surface charge and gel thickness (A) Signup and view all the answers

What can be inferred if the proteins are invisible after running electrophoresis?

They need to be stained for visibility (B) Signup and view all the answers

What happens to proteins with a negative charge when they migrate toward the anode?

They gain protons and become less negatively charged. (A) Signup and view all the answers

What is the destination for proteins during isoelectric focusing?

The point where the pH equals the protein's isoelectric point. (C) Signup and view all the answers

Why is calculating a protein's isoelectric point difficult in complex, folded proteins?

The pKa values of the side chains can fluctuate based on environment. (B) Signup and view all the answers

What is the primary advantage of using isoelectric focusing in protein analysis?

It directly measures the isoelectric point of proteins. (B) Signup and view all the answers

What is the purpose of combining isoelectric focusing with SDS-PAGE in two-dimensional electrophoresis?

To enhance the resolution of protein bands. (C) Signup and view all the answers

What is the first step when performing two-dimensional electrophoresis?

Isoelectric focusing of the sample. (B) Signup and view all the answers

In isoelectric focusing, what charge do proteins have when they arrive at their isoelectric point?

They have no net charge. (D) Signup and view all the answers

What role does SDS play in the two-dimensional electrophoresis process?

It charges proteins negatively for separation. (C) Signup and view all the answers

Study Notes

Gel Electrophoresis

Gel electrophoresis is a technique used to separate biomolecules based on size, charge, and other characteristics.
It involves generating an electric field to force molecules to migrate through a gel.
Electrophoresis methods are used to purify and characterize biomolecules (proteins, DNA, RNA).
Charged particles migrate through a gel (e.g., agarose, polyacrylamide) in an electric field.
Different proteins (DNA/RNA) migrate differently due to their varying abilities to move through the gel matrix.

Principles of Electrophoresis

Electrophoresis uses an electric field to move charged particles (biomolecules).
Separation is based on size and charge differences among particles.
The gel matrix acts as a sieve, impeding the movement of larger molecules.
Higher magnitude charges migrate faster across the gel.
Common gel materials are agarose (complex carbohydrate) and polyacrylamide (organic polymer).
Larger molecules migrate more slowly.

Electrophoresis Techniques

Electrophoresis, in general uses an electrolytic cell, causing reduction reactions to occur at the cathode.
The negatively charged electrode is the cathode and the positively charged electrode is the anode.
Negatively charged molecules migrate toward the anode.
Positively charged molecules migrate toward the cathode.
Smaller molecules migrate faster through the gel.
Samples are usually loaded near the cathode.
Visualizing Separated Molecules (Proteins)
Molecules aren't initially visible, so staining is needed.
Coomassie (binds to proteins, causing them to appear blue).
Fluorescent dye (e.g ethidium bromide) binds to nucleic acids, glows under UV light(orange).
Bands visualised with DNA/RNA (indicate molecule size and amounts).

Native Electrophoresis

Preservation of a molecule's structure and function.
Separation primarily based on size (DNA molecules migrate slower, larger molecules migrate slowly).
Circular/folded molecules migrate faster as they are more compact.
Used for DNA and RNA typically.

Denaturing Electrophoresis (SDS-PAGE)

Protein structure is disrupted.
SDS coats proteins providing a negative charge.
All proteins with SDS become negatively charged.
Proteins primarily separate by size(larger molecules migrate slower).
Used to separate proteins typically.
Separates by size because of uniform negative charge from SDS.
Proteins denatured (3D structures disrupted).

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Description

Test your knowledge on the principles of protein electrophoresis and the factors affecting protein migration in native and denaturing PAGE. This quiz covers key concepts such as the effects of charge, size, and pH on protein separation. Ideal for students studying biochemistry or molecular biology.