3. Electrophoresis

What is electrophoresis?

The migration of charged molecules in solution in response to an electric field.

What is gel electrophoresis?

The migration of charged molecules through a gel under electric current.

What is a primary purpose of gel electrophoresis?

To separate DNA, RNA, or protein molecules.

What type of gel is typically used in electrophoresis?

A gel is a Jello-like matrix with pores made of acrylamide or agarose.

What is a significant risk associated with electrophoresis laboratories?

High risk of electric shock.

What should be done to ensure laboratory safety in electrophoresis?

Use equipment with electrical interlocks and verify the integrity of the tank regularly.

What is an agarose gel made from?

Agarose, which is derived from seaweed.

How does the concentration of agarose affect DNA migration speed?

Higher agarose concentration means smaller pores, which slows down larger DNA fragments.

What is sodium dodecyl sulfate-polyacrylamide gel electrophoresis?

SDS-PAGE is a technique that denatures proteins and sorts them based on size.

What is the purpose of using molecular weight markers in gel electrophoresis?

To identify the size of molecules by comparing them against a size ladder.

What safety precautions should be taken when using polyacrylamide gels?

Handle with care as acrylamide is a neurotoxin.

What buffer is commonly used in serum protein electrophoresis?

Barbital buffer with a standard ionic strength and pH.

What does the term 'ionic strength' refer to in electrophoresis?

The concentration of ions in the buffer that conducts electric current.

What is Coomassie dye used for?

To stain proteins in gel electrophoresis for visualization.

What effect does high voltage have on agarose during electrophoresis?

Can cause heat degradation and may result in poor resolution.

What are the two types of polyacrylamide gels used in electrophoresis?

Non-denaturing and denaturing gels.

What is the main advantage of constant current in gel electrophoresis?

Maintains a constant migration rate for samples.

What is a drawback of constant voltage settings in electrophoresis?

Longer run times as current decreases.

Which type of electrophoresis allows for protein activity assays?

Blue Native PAGE.

What happens during electroendosmosis?

Positive ions from the buffer move towards the negative cathode, affecting macromolecule motion.

Why is it important to use a loading dye in gel electrophoresis?

To visualize sample migration and track its progress in the gel.

What is the purpose of immunoelectrophoresis?

To separate antigens and determine the types of antigens present.

How is 2D SDS-PAGE different from regular SDS-PAGE?

It separates proteins based on two properties: isoelectric point and mass.

What is the function of the buffer in gel electrophoresis?

To provide ions for current flow and maintain pH during the run.

What can cause smearing in gel electrophoresis results?

Sample overload and weak staining solutions.

What is the significance of having a constant power setting in electrophoresis?

Maintains consistent heat production, though migration rates can vary.

What is the effect of gel concentration on the separation of DNA fragments?

Higher concentration equals smaller pores, slow migration of larger fragments.

How is visualization attained for DNA/RNA in gel electrophoresis?

Using stains like Ethidium Bromide or SYBR Green.

What aspect of the gel affects DNA's rate of migration?

The size and conformation of the DNA molecules.

What role do sample buffers play in capillary electrophoresis?

They conduct current and maintain ionic strength.

What happens when the ionic strength of the buffer decreases during electrophoresis?

It increases resistance, affecting migration.

What additional benefit does gel-facilitated sieving provide in capillary electrophoresis?

Improves separation of DNA fragments based on size.

What should be done to a gel after running electrophoresis?

It should be fixed to immobilize proteins and stained for visualization.

What is the purpose of using a size ladder during gel electrophoresis?

To estimate the size of unknown samples.

What immune system conditions can be diagnosed through serum protein electrophoresis?

Conditions such as cancer or kidney problems.

What is the primary goal of serum protein electrophoresis?

To separate and analyze the various proteins in blood serum.

How does the conformation of DNA affect its migration speed?

Closed DNA migrates faster than circular, supercoiled, or linear forms.

What is the main advantage of capillary electrophoresis?

Higher resolution and smaller sample volumes.

How can ionic strength be compensated in electrophoresis?

By ensuring the buffer and gel have matching ionic strengths.

What can result from inappropriate buffer pH during protein electrophoresis?

Unexpected migration or distorted bands.

What characteristic of agarose makes it suitable for electrophoresis?

It is non-toxic and easy to prepare.

For what purpose is gel red used in gel visualization?

To stain DNA; it is non-toxic and non-mutagenic.

What phenomenon aids in separating proteins in immunofixation electrophoresis?

The binding of antibodies to specific antigens.

Why might one choose agarose gel electrophoresis over polyacrylamide?

It is easier to prepare and less toxic.

What are the potential consequences of using the incorrect staining solution?

Poor visualization and inaccurate results.

What does the 'pH gradient' in 2D SDS-PAGE help achieve?

Separation of proteins based on their isoelectric point.