Biochemistry: Protein Electrophoresis Techniques. 9

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Questions and Answers

Why is SDS-PAGE performed under denaturing conditions?

To facilitate the separation of proteins by their charge-to-mass ratio.

To ensure that proteins migrate based solely on their molecular weight. (correct)

To enhance the binding of SDS to the protein molecules.

To prevent protein aggregation during electrophoresis.

What is the primary function of SDS in SDS-PAGE?

To act as a catalyst for protein digestion during electrophoresis.

To stain the protein molecules during electrophoresis.

To provide a negatively charged backbone to the protein molecules. (correct)

To act as a reducing agent and break disulfide bonds.

What is the role of reducing agents like beta-mercaptoethanol or DTT in SDS-PAGE?

To prevent the formation of disulfide bonds between proteins.

To neutralize the negative charge of SDS and ensure proper protein migration.

To enhance the binding of SDS to protein molecules.

To break disulfide bonds within proteins, allowing for complete denaturation. (correct)

Which of the following statements accurately explains the function of BSA in the context of biochemical applications?

BSA serves as a neutral, non-reactive protein, providing a stable environment for reactions. (B) Signup and view all the answers

Which of the following is NOT a direct application of SDS-PAGE as described in the text?

Investigating the interaction between proteins. (D) Signup and view all the answers

What is the significance of separating proteins in the fetal bovine serum (FBS) by SDS-PAGE?

Analyzing the composition of proteins in the serum for quality control. (D) Signup and view all the answers

Which of the following statements accurately describes the role of SDS in enhancing protein staining?

SDS disrupts the protein structure, creating more binding sites for the stain. (C) Signup and view all the answers

If a protein sample is subjected to SDS-PAGE without the addition of a reducing agent, how would the results differ from those obtained with a reducing agent?

Proteins with disulfide bonds would migrate as a single band, representing the intact protein. (B) Signup and view all the answers

Why is fetal bovine serum (FBS) considered a by-product of the meat/dairy industry?

FBS is collected from the blood of calves during the production of meat and dairy products. (B) Signup and view all the answers

Which of the following scenarios would NOT be suitable for using SDS-PAGE as a technique?

Identifying specific DNA sequences within a sample. (B) Signup and view all the answers

What is the total volume of the sample in well 2 when combining the components?

40 µl (A) Signup and view all the answers

Which well contains the highest total mass of the marker/sample?

Well 5 (A) Signup and view all the answers

In well 4, how much protein is added to the sample preparation?

2 µl (D) Signup and view all the answers

What is the volume of buffer used in well 7?

10 µl (B) Signup and view all the answers

How does the total mass of the marker/sample in well 8 compare to the ones in other wells?

It is the least among all wells. (C) Signup and view all the answers

What is the primary purpose of using polyacrylamide gel in protein electrophoresis?

To allow for the separation of protein molecules based on size (C) Signup and view all the answers

Which agent is commonly used as a cross-linking agent in the preparation of polyacrylamide gels?

N,N-methylenebisacrylamide (A) Signup and view all the answers

What determines the resultant electric charge of a protein molecule during electrophoresis?

The pH of the solution (A) Signup and view all the answers

What is the result of the ionization of carboxyl and amino groups in amino acids?

They gain additional electric charge (A) Signup and view all the answers

How is the strength of the electric field relevant to protein electrophoresis?

It affects the intensity with which proteins interact with the electric charge. (B) Signup and view all the answers

What characteristic of proteins allows them to exhibit both basic and acidic properties?

The amphoteric nature of amino acid residues (B) Signup and view all the answers

What is the significance of the isoelectric point (pI) for different proteins?

It is the pH at which proteins are least soluble. (B) Signup and view all the answers

What is a main concern when handling acrylamide in its monomeric form?

It is a potent neurotoxin (C) Signup and view all the answers

What is the primary purpose of using SDS in protein electrophoresis?

To mask the original charges of proteins (A) Signup and view all the answers

How does the concentration of acrylamide affect protein separation in gel electrophoresis?

Lower concentrations allow for the separation of low molecular weight proteins (D) Signup and view all the answers

What role does N,N-methylenebisacrylamide play in the preparation of polyacrylamide gels?

It serves as a cross-linking agent (D) Signup and view all the answers

What characteristic of proteins does SDS-PAGE electrophoresis use for separation under denaturing conditions?

Differences in protein mass (C) Signup and view all the answers

What factor does NOT influence the migration rate of proteins in polyacrylamide gel electrophoresis?

The presence of reducing agents (B) Signup and view all the answers

What is the significance of the chemical initiator ammonium persulfate in the polymerization process?

It promotes the polymerization reaction of acrylamide (A) Signup and view all the answers

What effect does the cross-linking density have in protein electrophoresis?

It influences the migration rate of proteins (B) Signup and view all the answers

Which statement is true regarding the migration of polypeptides during SDS-PAGE?

Migration is influenced by the charge-to-mass ratio (D) Signup and view all the answers

What is a potential health hazard associated with the use of acrylamide in gel preparation?

It remains a neurotoxin even after polymerization (C) Signup and view all the answers

Why is it important to ensure denaturing conditions during protein electrophoresis?

To ensure uniform physicochemical properties across protein molecules (C) Signup and view all the answers

What is one primary function of bovine serum albumin (BSA) in biochemical procedures?

To block unoccupied binding surfaces on substrates (B) Signup and view all the answers

Which of the following correctly describes the role of BSA in enzyme reactions?

It stabilizes the enzyme functions (D) Signup and view all the answers

Which of the following is NOT a component of the running buffer for SDS-PAGE?

BSA (A) Signup and view all the answers

What is the mass range of the new protein molecular weight marker used in SDS-PAGE?

10 to 250 Da (D) Signup and view all the answers

How should the 10x electrophoresis buffer be prepared for use in SDS-PAGE?

Dilute with 800 ml of ddH2O (B) Signup and view all the answers

Which group of proteins is primarily found in milk?

Caseins (A) Signup and view all the answers

What is the purpose of adding beta-mercaptoethanol to the Laemmli buffer in SDS-PAGE?

To reduce disulfide bonds (A) Signup and view all the answers

Which substrate's role is to prevent adherence of contaminants in biochemical assays?

Bovine serum albumin (BSA) (A) Signup and view all the answers

What does milk composition include among its five main groups of compounds?

Lipids (A), Sugars (C) Signup and view all the answers

In the context of SDS-PAGE, what does diluting FBS, BSA, and other solutions involve?

Combining with ddH2O as per a specified table (D) Signup and view all the answers

Study Notes

Protein Electrophoresis via SDS-PAGE

Electrophoresis separates molecules based on their movement in an electric field due to uneven charge.
Polyacrylamide gel electrophoresis is commonly used for protein separation, allowing separation of proteins between 5 kDa and 300 kDa.
Separates polynucleotides sized 5-2,000 base pairs, stained with silver for visible and lasting images.
Cost-effective method.

Polyacrylamide Gels

Prepared from acrylamide monomers and cross-linking agents (like bis-acrylamide).
Polymerization is a free-radical reaction, initiated chemically or photochemically.
Cross-linking density and pore size are controlled by acrylamide and bis-acrylamide concentrations.
Acrylamide is a neurotoxin.

Protein Properties

Amino acids, peptides, and proteins are amphoteric (amphiprotic/amphoteric), acting as both acids and bases.
Net positive or negative charge depends on environmental conditions.
The amphoteric characteristic results from amino acid residues behaving as both basic and acidic.
Charge depends on pH-dependent ionization of carboxyl and amino groups.

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

Denaturing conditions (buffers, gels, voltages) ensure uniform physicochemical properties of the protein molecules.
Separation based solely on differences in protein mass.
Proteins with higher molecular weights migrate slower and proteins with lower molecular weights migrate faster.
Spatial structures (secondary, tertiary, quaternary) and charges, affect migration speed.
SDS binds to proteins (forming protein-SDS complexes), giving proteins a negative charge.
Ratio of electric charge to mass is fixed, causing proteins to migrate towards positive anode.
Amount of bound SDS is proportional to protein size (1g of protein binds 1.4g of SDS).
Protein spatial structures unfold (denature).
Eliminates enzymatic protein degradation.
Efficient staining of SDS-protein complexes compared to staining proteins alone.
Covalent bonds (disulfide bridges) are broken using reducing agents (mercaptoethanol or DTT).

SDS-PAGE Method

Allows for precise determination of protein molecular weights.
Identifies and monitors protein mixtures' composition.
Checks protein homogeneity
Analyzes subunits of biologically active protein complexes.

Practical Applications (Examples of Protein Samples)

Fetal Bovine Serum (FBS): Used in cell cultures, obtained from fetal calves.
Bovine Serum Albumin (BSA): Neutral, non-reactive protein in biochemistry and molecular biology.
Milk (animal-origin & non-animal origin): Contains proteins like casein, albumin, and immunoglobulins.

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Description

Explore the principles and techniques of protein electrophoresis via SDS-PAGE in this quiz. Learn about polyacrylamide gel preparation, protein properties, and the significance of separation methods in biochemistry. Test your knowledge of these essential laboratory techniques.