SDS-PAGE and Protein Electrophoresis

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Questions and Answers

What is the primary purpose of SDS-PAGE?

To purify nucleic acids

To determine amino acid sequences

To separate proteins based on their molecular weights (correct)

To separate proteins based on their charge

What does SDS stand for in the context of SDS-PAGE?

Sulfuric Dodecyl Solution

Sodium Dodecyl Sulfate (correct)

Sodium Decyl Sulfate

Sodium Dodecyl Sulfide

What is the role of the detergent SDS in electrophoresis?

To increase the pH of protein samples

To ensure all proteins have a similar charge (correct)

To provide an electric current

To degrade proteins for separation

Which component is the gel made of in SDS-PAGE?

Acrylamide polymer Signup and view all the answers

What does PAGE stand for in the SDS-PAGE technique?

Polyacrylamide Gel Electrophoresis Signup and view all the answers

Which factor influences the migration of proteins during electrophoresis?

The molecular weight of the proteins Signup and view all the answers

Why might some proteins not migrate during electrophoresis?

If they lack a charge Signup and view all the answers

What is one application of SDS-PAGE?

Assessing the purity of protein samples Signup and view all the answers

What is the primary purpose of denaturing proteins with SDS in electrophoresis?

To ensure they lose their secondary, tertiary, or quaternary structure Signup and view all the answers

How does SDS affect the charge of proteins during electrophoresis?

It gives them a uniform negative charge Signup and view all the answers

Which of the following structures does SDS denaturation primarily target?

Secondary and tertiary structures Signup and view all the answers

What does the migration of proteins in an electric field during electrophoresis depend on?

The size of the protein Signup and view all the answers

Which gel type has larger pores and therefore is less effective for separating proteins by size?

Agarose gel Signup and view all the answers

What is typically used to visualize proteins after electrophoresis?

Protein-specific staining techniques Signup and view all the answers

What effect does protein structure have on migration during electrophoresis?

It can cause proteins to migrate faster than their size suggests Signup and view all the answers

What is a consequence of noting the migration distance of proteins during electrophoresis?

It permits size estimation compared to a known molecular weight marker Signup and view all the answers

What is the role of glycerol in the SDS-PAGE loading buffer?

To increase the density of the sample Signup and view all the answers

Which component of the SDS-PAGE process is responsible for tracking the migration of samples?

Bromophenol blue Signup and view all the answers

What characteristic of the stacking gel distinguishes it from the resolving gel?

Different pH level Signup and view all the answers

What is the primary purpose of the protein ladder loaded into one of the wells?

To provide a standard for molecular weight determination Signup and view all the answers

Which of the following buffers is NOT used in the SDS-PAGE process?

Cooling buffer Signup and view all the answers

What happens when proteins are treated with SDS in the preparation stage of SDS-PAGE?

Proteins are denatured and acquire negative charge Signup and view all the answers

What is the primary purpose of sodium dodecyl sulfate (SDS) in SDS-PAGE?

To denature proteins and eliminate structural influences Signup and view all the answers

At what temperature is the protein sample typically heated during SDS-PAGE preparation?

95°C Signup and view all the answers

At what pH are the resolving gels typically maintained during SDS-PAGE?

Around 8.8 Signup and view all the answers

How does the SDS-PAGE process ensure the separation of proteins based on size?

By the formation of a dense gel matrix Signup and view all the answers

What is the function of a reducing agent like β-mercaptoethanol (BME) in SDS-PAGE?

To cleave disulfide bridges in proteins Signup and view all the answers

What is the typical molecular weight range that the vertical electrophoresis system can separate?

5 - 250 kDa Signup and view all the answers

What is the purpose of the stacking gel in SDS-PAGE?

To concentrate proteins before they enter the resolving gel Signup and view all the answers

What prevents air bubbles from forming in the resolving gel during its preparation?

Layering isopropanol over the gel Signup and view all the answers

Which component significantly contributes to the formation of the polyacrylamide gel matrix?

Acrylamide monomer Signup and view all the answers

Why is the influence of protein structure and charge eliminated during SDS-PAGE?

SDS provides a uniform charge-to-mass ratio to proteins Signup and view all the answers

Study Notes

SDS-PAGE

SDS-PAGE is a technique used to separate proteins based on their molecular weights
Proteins are negatively charged in SDS-PAGE, meaning they migrate towards the positive electrode
SDS (Sodium Dodecyl Sulfate) denatures proteins and gives them a uniform negative charge, eliminating the effect of charge and structure
Proteins are separated solely based on differences in their molecular weights
Molecular weight of a protein is the sum of masses of its constituent amino acids
The separation is achieved within a polyacrylamide gel with a mesh size that is suitable for the separation of proteins of typical size.

Protein Electrophoresis

Electrophoresis is a technique that separates macromolecules (proteins) in a mixture using an electric current
In this process, proteins migrate through the gel, separated by their molecular weight
The polyacrylamide gel matrix acts as a molecular sieve, with smaller proteins moving through the pores faster than larger proteins
SDS-denatured proteins migrate based on their size rather than other factors.

Applications of SDS-PAGE

Protein Purification: Evaluating the purity of protein samples
Molecular Weight Estimation: Determining the protein's molecular weight
Protein Identification: Identifying proteins in complex mixtures
Comparative Analysis: Comparing the expression levels of proteins under different conditions

Preparing Proteins for SDS-PAGE

Denaturing proteins, before electrophoresis, is often necessary for a conclusive result
Heat destroys hydrogen bonds while reducing agents, such as $\beta$-mercaptoethanol (BME), break disulfide bonds
This linearizes the proteins, making them uniform, and only size-dependent

Materials Required for SDS-PAGE

Gel apparatus and electrophoresis apparatus
30% acrylamide/0.8% bis-acrylamide stock solution
2.5x separating gel buffer
5x stacking gel buffer
TEMED (Tetramethylethylenediamine)
10% ammonium persulfate
Butane-1,2-diol
5x electrophoresis buffer
5x SDS sample buffer
Protein samples
Protein Ladder (for size determination)

Preparation of Polyacrylamide Gel

Gel casting
Resolving gel
Stacking gel
Isopropanol is placed atop the resolving gel to prevent air bubbles and make the resolving gel surface smoother
The stacking gel has lower acrylamide percentage, different pH and ionic composition compared to the resolving gel
A comb is placed atop the gel to create “wells” where protein samples can be loaded
Different amounts of acrylamide and bis-acrylamide form gels with varying pore sizes suitable for different protein sizes

Run the SDS-PAGE

After casting the gel, samples are loaded into wells
An electric current is passed through the gel, causing the negatively charged proteins to migrate towards the positive electrode

Running Buffer

Maintains the correct pH, facilitates the current flow and electrophoretic separation
Three buffers are usually used in SDS-PAGE: buffer for casting the gel, buffer for sample preparation and buffer for running the gel

Loading Samples

Each sample is loaded into a well in the gel, using SDS-PAGE loading buffer
The loading buffer contains SDS and usually also glycerol (this increases the density of the sample) and a tracking dye such as bromophenol blue, which helps to monitor the migration of the sample through the gel
Samples should be heated prior to loading to ensure proteins are denatured and linear

Glycerol

Increases the density of the sample so the sample drops to the bottom of the well, preventing spillover

Bromophenol Blue

Dye used to track the migration of the sample, and thus can be used to determine migration distances
The dye is negatively charged and will migrate towards the positive electrode, helping determine when the separation is complete

Protein Ladder

Contains protein samples of known molecular weights
Used to determine the estimated size/weight of the unknown samples
Each rung of the ladder corresponds to a polypeptide of a specific size.

Visualization of the Gel

Coomassie Blue Staining- Visualizing and identifying separated protein bands
Silver Staining- Sensitive, able to detect as little as 2-5ng of protein per band
Gels are incubated in a solution that stains the separated proteins, making them visible

Interpreting Results

The separated proteins will form bands in the gel
The distance traveled by a protein can be used to estimate its molecular weight
Comparing the migration distance of the sample protein to the known molecular weight markers in the protein ladder can determine the protein’s size

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Description

This quiz explores the principles and techniques of SDS-PAGE, a method used to separate proteins based on molecular weight. It covers the role of sodium dodecyl sulfate, the process of electrophoresis, and how proteins are separated in a polyacrylamide gel. Test your knowledge on these key concepts in protein analysis!