Biochemical Techniques: SDS-PAGE

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Questions and Answers

What is the primary purpose of SDS-PAGE electrophoresis?

To separate proteins by their size (correct)

To separate proteins by their charge

To stain proteins for visualization

To separate nucleic acids by their sequence

What component is primarily responsible for providing a uniform negative charge to polypeptides in SDS-PAGE?

Polyacrylamide gel

Gel pore size

Electric field

Sodium Dodecyl Sulfate (SDS) (correct)

How does the pore size of the gel affect the SDS-PAGE results?

It determines the voltage applied

It affects the resolution of the DNA bands

It influences the mobility of proteins (correct)

It modifies the staining process

In SDS-PAGE, where do the protein bands arrange based on their molecular weight?

From cathode to anode Signup and view all the answers

What factors can influence the rate of movement of proteins during SDS-PAGE?

Gel pore size and strength of electric field Signup and view all the answers

What is a common application of SDS-PAGE besides separating proteins?

Separating DNA and RNA molecules Signup and view all the answers

What type of power supply is typically needed for running SDS-PAGE?

100-200 volts Signup and view all the answers

Which of the following is not typically part of the SDS-PAGE instrumentation?

Fluorescent lighting system Signup and view all the answers

What role does acrylamide play in SDS-PAGE electrophoresis?

It is the supporting medium that polymerizes to form polyacrylamide. Signup and view all the answers

What defines the resolving gel in SDS-PAGE electrophoresis?

It typically contains 5-10% acrylamide. Signup and view all the answers

Which statement correctly describes the stacking gel in SDS-PAGE?

It constitutes 1/3rd of the gel plate and contains 2-5% acrylamide. Signup and view all the answers

Why are different buffers used for the upper and lower reservoirs in SDS-PAGE?

To ensure different pH levels in the stacking and running gels. Signup and view all the answers

What is the primary function of Comassie Brilliant Blue R-250 in SDS-PAGE?

To detect proteins after separation by creating visible blue bands. Signup and view all the answers

What happens to the excess dye incorporated in the gel after staining?

It is removed by a specific de-staining solution. Signup and view all the answers

How does the percentage of acrylamide in the stacking gel affect protein identification?

Is inversely related to the size of the protein to be identified. Signup and view all the answers

What is the main property of the resolving gel that assists in SDS-PAGE?

It has smaller diameter pores that impact the sieving property. Signup and view all the answers

What is the primary role of SDS in SDS-PAGE electrophoresis?

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

Which of the following best describes reducing SDS-PAGE?

It involves the use of heat and a reducing agent to denature proteins. Signup and view all the answers

What occurs to proteins as they migrate through the SDS-PAGE gel?

Short proteins find it easier to fit through the gel pores than larger ones. Signup and view all the answers

After electrophoresis, how are the separated protein bands visualized?

Through the use of colored dyes that bind to proteins Signup and view all the answers

Which of the following is NOT an application of SDS-PAGE?

Gene cloning Signup and view all the answers

What advantage does SDS-PAGE offer over native PAGE?

It allows all proteins to migrate towards the anode. Signup and view all the answers

During the SDS-PAGE process, what is the purpose of boiling the protein mixture?

To further denature proteins and reduce disulfide linkages Signup and view all the answers

Which statement correctly describes the separation process in SDS-PAGE?

Proteins separate based on their size as they navigate the gel matrix. Signup and view all the answers

Study Notes

SDS-PAGE Electrophoresis

Sodium Dodecyl Sulfate (SDS) polyacrylamide gel electrophoresis separates proteins based on size and molecular weight.
Anionic detergent SDS denatures proteins into polypeptide subunits, imparting a uniform negative charge.
Uniform charge allows polypeptides to migrate solely based on molecular mass, eliminating shape impacts on movement.
Denatured proteins loaded at the cathode migrate through polyacrylamide gel, producing clear bands in order of decreasing molecular mass.

Instrumentation

Electrophoretic apparatus includes a vertical or horizontal tank, electrodes, gel cassettes, Teflon spacers, clips, and pipettes.
A power supply ranging from 100-200 volts powers the protein resolving gels.
Staining boxes, typically made of clear plastic, are resistant to organic dyes for staining gels post-electrophoresis.

Supporting Media

Acrylamide serves as the supporting medium, creating a polyacrylamide gel via polymerization.
Two types of gels are used:
- Resolving Gel (or Running Gel): About 5-10% acrylamide, constituting 2/3 of the gel, provides smaller pore sizes for sieving proteins.
- Stacking Gel: About 2-5% acrylamide, constituting 1/3 of the gel, features larger pores to facilitate sample concentration before entering the resolving gel.

Buffers

Amine buffers are used in both gel reservoirs; lower reservoir adjusts using HCl and upper with glycine for optimal pH differences.

Staining

Coomassie Brilliant Blue R-250 (CBB) is a widely used protein stain, binding non-specifically to proteins.
Fixed proteins in the gel are stained with CBB and excess dye is removed via a de-staining process to reveal blue bands.

Procedure

Protein samples are mixed with SDS for denaturation, optionally boiled with reducing agents (e.g., DTT or 2-mercaptoethanol) to disrupt tertiary and quaternary structures.
Denatured proteins are loaded onto stacking gel wells, connected to the cathode.
Upon applying current, proteins migrate towards the anode through stacking and then running gel matrices, separating based on size.
Post-separation, gels are stained and de-stained to visualize protein bands for analysis.

Applications

Establishing protein size and identity.
Determining sample purity and protein quantification.
Useful in blotting applications.

Advantages of SDS-PAGE

High molecular mobility and rapid separation due to uniform negative charge on proteins.
SDS-treated proteins provide more consistent results than native proteins.

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Description

Explore the principles and applications of SDS-PAGE in the context of chromatography and electrophoresis. This quiz covers the essential techniques used in separating proteins based on size, focusing on the key concepts presented in Unit III of BBT-304. Test your understanding of this powerful biochemical method!