SDS-PAGE Electrophoresis Overview

Questions and Answers

What is the primary role of the stacking gel in SDS-PAGE?

To separate proteins based on their size.

To compress proteins into a narrow band for more efficient separation. (correct)

To neutralize the charge of proteins before separation.

To increase the overall conductivity of the gel.

Which component of the gel primarily influences the mobility of proteins during electrophoresis?

The presence of salt in the buffer.

The charge of the proteins.

The pH of the resolving gel. (correct)

The thickness of the gel.

What is the effect of SDS on proteins during electrophoresis?

It eliminates the primary structures of the proteins.

It provides a constant charge to mass ratio across different proteins. (correct)

It enhances the native conformation of proteins.

It increases the overall mass of the proteins.

In a discontinuous gel system, what role does the buffer in the lower chamber play?

It drives the proteins through the gel during separation. Signup and view all the answers

Which of the following statements about the resolving gel is accurate?

It contributes to the separation of proteins mainly based on size. Signup and view all the answers

How does the mobility of proteins change when the ionic front reaches the resolving gel?

Mobility increases due to a change in the ionic environment. Signup and view all the answers

What is the significance of the different buffers in a discontinuous gel system?

They create distinct electrochemical environments for effective separation. Signup and view all the answers

Which ions are used in the stacking gel to help compress the proteins?

Glycinate and chloride. Signup and view all the answers

What is the primary function of sodium dodecyl sulphate (SDS) in SDS-PAGE?

To denature proteins and impart a uniform charge Signup and view all the answers

How do the charges of proteins affect their movement in an electric field during electrophoresis?

Proteins vary in charge and migrate accordingly Signup and view all the answers

What is the purpose of using reducing agents like DTT in SDS-PAGE?

To reduce disulphide bonds within and between proteins Signup and view all the answers

What distinguishes discontinuous gels from continuous gels in electrophoresis?

Discontinuous gels allow for different migration rates in different gel layers Signup and view all the answers

What role does the stacking gel play in SDS-PAGE?

It concentrates the proteins into a narrow band before they enter the separating gel Signup and view all the answers

Why is separation of DNA fragments considered simpler than separation of proteins?

DNA is always negatively charged regardless of size Signup and view all the answers

What is the result of using SDS on multi-subunit proteins during SDS-PAGE?

They are denatured and separated into individual polypeptide chains Signup and view all the answers

What is a primary challenge in separating a mixture of solubilised proteins?

The varying overall net charge of proteins complicates their migration Signup and view all the answers

What is the purpose of DTT in protein sample preparation prior to SDS-PAGE?

To break disulphide linkages Signup and view all the answers

Why is the polymerization of polyacrylamide crucial for SDS-PAGE?

It forms channels for protein separation Signup and view all the answers

What role does methylene bisacrylamide play in gel preparation?

It cross-links polymer chains Signup and view all the answers

How does SDS contribute to the electrophoresis process?

By providing a uniform charge-to-mass ratio to proteins Signup and view all the answers

What is the main function of the stacking gel in SDS-PAGE?

To concentrate the proteins into a narrow band before they enter the separating gel Signup and view all the answers

What factor can be altered to change the pore size of the separating gel?

The amount of methylene bisacrylamide used Signup and view all the answers

What is the primary benefit of using TEMED in gel preparation?

To catalyze the polymerization reaction Signup and view all the answers

In the context of electrophoresis, what does the sieving effect refer to?

The ability of the gel to restrict larger molecules Signup and view all the answers

Study Notes

Electrophoresis in SDS-PAGE

SDS (sodium dodecyl sulfate) imparts a uniform negative charge to proteins, enabling their flow towards the anode during electrophoresis.
Two buffer chambers exist in a protein gel system: an upper chamber for stacking gel and a lower chamber for resolving gel.
Different buffers create distinct electrochemical environments, facilitating protein movement into and through the gel.
The stacking gel focuses proteins, while the resolving gel separates them based on size due to smaller pore size.

Buffer Dynamics

Chloride ions (green) and glycinate ions (orange) compress proteins before entering the resolving gel.
At a pH of 8.3 in the resolving gel, increased ion mobility enhances protein mobility, with smaller proteins moving faster than larger ones.
Stacking gel pH is typically set at 6.7, while the resolving gel maintains pH 8.3, optimizing protein separation.

SDS-PAGE Overview

SDS-PAGE is crucial for separating heterogeneous protein mixtures based on size and charge.
Proteins' negative charges vary; hence, a uniform charge needs to be imparted for effective separation.
SDS denatures proteins, providing a net negative charge proportional to their size, allowing for size-based migration during electrophoresis.

Sample Preparation

The application of powerful reducing agents (e.g., DTT or mercaptoethanol) is essential for breaking disulfide bonds in proteins.
Heating samples near 100°C ensures complete denaturation of proteins when combined with SDS and reducing agents.

Gel Composition

Polyacrylamide gels are hydrophilic and tough, requiring cross-linking of polymer chains for effective sieving during electrophoresis.
Methylene bisacrylamide serves as the crosslinker in gel formation, creating pores for protein separation.
Variations in the amount of the crosslinker influence pore size, thus determining the range of protein sizes that can be effectively separated.

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Description

This quiz covers the principles and dynamics of SDS-PAGE, focusing on how proteins are separated based on size and the role of various buffer systems. Learn about the importance of pH and ion mobility in optimizing protein separation during electrophoresis. Test your understanding of this vital biochemical technique.