Biochemistry: SDS-PAGE and Gel Techniques 7

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Questions and Answers

What is the primary purpose of using polyacrylamide gel electrophoresis (PAGE) in protein analysis?

To determine the molecular weight of proteins by size separation. (correct)

To analyze the amino acid sequence of proteins.

To study the three-dimensional structure of proteins.

To identify specific protein interactions within a sample.

What is the role of N,N-methylenebisacrylamide (bis-acrylamide) in polyacrylamide gel electrophoresis?

Acts as a reducing agent to break disulfide bonds in proteins.

Serves as a buffer to maintain the pH of the electrophoresis solution.

Provides a cross-linking agent to control gel pore sizes. (correct)

Initiates the polymerization reaction of acrylamide monomers.

Why is the use of acrylamide in its monomeric form considered potentially hazardous?

Acrylamide readily binds to DNA, interfering with gene expression.

Acrylamide reacts with proteins, altering their structure and function.

Acrylamide is a strong oxidizing agent, causing cell damage.

Acrylamide is a potent neurotoxin, affecting the nervous system. (correct)

What property of amino acids allows proteins to act as both bases and acids, exhibiting amphoteric behavior?

The presence of a carboxyl group (-COOH) and an amino group (-NH2) in their structure. (A) Signup and view all the answers

How does the isoelectric point (pI) of a protein relate to its net electric charge?

The pI is the pH at which the protein has a net zero charge. (C) Signup and view all the answers

In SDS-PAGE, what is the purpose of adding sodium dodecyl sulfate (SDS) to the protein samples?

To create a uniform negative charge on all protein molecules. (C) Signup and view all the answers

Which of the following is NOT a benefit of using polyacrylamide gel electrophoresis for separating proteins?

Ability to analyze protein interactions within a complex mixture. (B) Signup and view all the answers

What is the main reason why polyacrylamide gel electrophoresis is commonly used for separating DNA fragments?

The gel provides a high resolution separation of DNA fragments based on their length. (B) Signup and view all the answers

What is the primary function of Sodium Dodecyl Sulfate (SDS) in SDS-PAGE?

To bind to proteins, giving them a uniform negative charge and promoting denaturation. (A) Signup and view all the answers

Why is the amount of SDS bound to a protein proportional to its size?

Larger proteins have a higher surface area, allowing for more SDS molecules to attach. (A) Signup and view all the answers

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

To break disulfide bonds, ensuring complete denaturation of proteins. (D) Signup and view all the answers

How does SDS-PAGE contribute to the accurate determination of protein molecular weights?

By using a standard curve to calculate the molecular weight based on the migration distance. (B) Signup and view all the answers

Which of the following is NOT a benefit of using SDS in SDS-PAGE?

Selective separation of proteins based on their biological activity. (D) Signup and view all the answers

What is the primary reason for denaturing proteins using SDS in SDS-PAGE?

To separate proteins based solely on their size, eliminating the influence of charge. (D) Signup and view all the answers

Why is it important to sterilize, filter, and perform quality control on Fetal Bovine Serum (FBS) used in cell cultures?

All of the above. (D) Signup and view all the answers

What is the primary purpose of conducting SDS-PAGE? Select the most comprehensive answer.

To analyze the composition of complex protein mixtures. (D) Signup and view all the answers

Which of the following statements accurately describes the migration of proteins in SDS-PAGE?

Proteins migrate towards the anode, due to the negative charge imparted by SDS. (C) Signup and view all the answers

What is the role of the polyacrylamide gel in SDS-PAGE?

To provide a porous matrix through which proteins can migrate based on their size. (C) Signup and view all the answers

What is the correct dilution ratio for the 10x electrophoresis buffer with double-distilled water?

1:10 (A) Signup and view all the answers

Which component is mixed with the Laemmli buffer containing beta-mercaptoethanol for sample preparation?

BSA (D) Signup and view all the answers

What is the thermal denaturation temperature and duration for the prepared samples?

99°C for 10 minutes (D) Signup and view all the answers

In preparing sample number 2 (FBS), what is the volume of water used?

1.5 µl (C) Signup and view all the answers

Which of the following is NOT listed as a reagent for the SDS-PAGE procedure?

Ethidium bromide (D) Signup and view all the answers

What is the primary function of BSA in biochemical protocols?

To block unoccupied binding sites on substrates (A) Signup and view all the answers

Which of the following proteins is NOT typically found in milk?

Bovine Serum Albumin (A) Signup and view all the answers

What characteristic of BSA makes it suitable for use in molecular biology?

It is a neutral, non-reactive protein (B) Signup and view all the answers

In terms of molecular weight, how does casein compare to other milk proteins?

It is the largest protein in milk (C) Signup and view all the answers

Which of the following functions is NOT associated with BSA in laboratory settings?

Acting as the primary nutrient for bacterial growth (B) Signup and view all the answers

What is the mass range of casein found in milk?

100-150 million Da (D) Signup and view all the answers

Which component is NOT one of the main groups found in milk?

Enzymes (C) Signup and view all the answers

What role does BSA play in relation to oxidizing agents during enzyme storage?

Minimizes the activity of oxidizing agents (A) Signup and view all the answers

Which of the following is a primary protein source in human nutrition derived from milk?

Casein (D) Signup and view all the answers

Which of these statements about immunoglobulins in milk is true?

They provide nutritional support to young mammals (D) Signup and view all the answers

What is the primary factor that influences the migration rate of proteins during electrophoresis?

The shape, charge, and mass of the protein (B) Signup and view all the answers

Which agent is commonly used to initiate the polymerization of acrylamide gels?

Ammonium persulfate (C) Signup and view all the answers

How does the cross-linking density of acrylamide gels affect protein migration?

Lower density gels are used for larger proteins while higher density gels are for smaller ones. (D) Signup and view all the answers

What is the primary purpose of sodium dodecyl sulfate (SDS) in protein electrophoresis?

To form protein-SDS complexes with a fixed charge-to-mass ratio (D) Signup and view all the answers

What characteristic of proteins can affect their migration through polyacrylamide gel besides mass?

The spatial structure of the proteins (A) Signup and view all the answers

Which variable is NOT directly adjustable in the preparation of polyacrylamide gels?

Temperature of the laboratory (D) Signup and view all the answers

What may pose a serious health hazard during the use of polyacrylamide gels?

Residual acrylamide monomers (D) Signup and view all the answers

The migration rate of polypeptides in gel electrophoresis can be primarily influenced by their:

Shape and charge (D) Signup and view all the answers

What occurs to proteins when treated with SDS?

They form complexes with a fixed ratio of charge to mass. (A) Signup and view all the answers

In protein electrophoresis, why are proteins with a higher molecular weight more likely to migrate slower?

They are physically larger and face more resistance. (C) Signup and view all the answers

Study Notes

SDS-PAGE Electrophoresis

Electrophoresis separates molecules based on their uneven electric charge, with polyacrylamide gel as the carrier for protein separation.
This method separates proteins ranging from 5 kDa to 300 kDa.
It can also separate polynucleotides sized 5-2,000 base pairs.
Staining with silver enables visible and lasting images.
Cost-effective.

Polyacrylamide Gels

Gels are made from acrylamide monomers and cross-linking agents (N,N-methylenebisacrylamide).
Polymerization is a free-radical reaction, often chemically or photochemically initiated.
Cross-linking density/pore size controls the separation process.
Acrylamide is a potent neurotoxin.

Amino Acid Properties

Amino acids, peptides, and proteins are amphoteric (amphiprotic/amphoteric).
This means they can act as both acids and bases, displaying a net positive or negative charge depending on environmental factors.
The charge arises from the ionization of side chains (carboxyl groups, amino groups) at varying pH levels.
Each protein has an isoelectric point where its total charge is zero.

Protein Electrophoresis Conditions

Denaturing conditions (buffers, gels, voltages) standardize protein properties for mass-based separation.
Proteins migrate through polyacrylamide based on size and charge.
SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) with SDS is a common separation method.
SDS creates protein-SDS complexes with consistent charge-to-mass ratio.
The complexes migrate toward the positive pole.
SDS's binding amount is proportional to the protein size.
SDS denatures proteins, unfolds polypeptide chains, and reduces enzyme activity during the separation process.
Disulfide bridges can be reduced with chemicals like mercaptoethanol/DTT.

SDS-PAGE Method

The SDS-PAGE method can perform accurate determination of separated protein molecular weights, monitor the composition of protein mixtures, check protein homogeneity, and analyze protein complex subunits.

Protein Samples for SDS-PAGE

FBS (Fetal Bovine Serum)
BSA (Bovine Serum Albumin)
Milk (animal and non-animal)
Protein power drink
Markers with known molecular weights (e.g., 250, 150,100, 75, 50, 37, 25, 15, 10 kDa)

Procedure

Dilute 10x electrophoresis buffer with distilled water.
Dilute protein samples and mix with Leammli buffer containing beta-mercaptoethanol.
Prepare sample solutions (specified volumes of protein, water, etc.) for wells on the gel.
Incubate samples in thermal block at 99°C for 10 minutes.
Set up electrophoresis apparatus, add appropriate samples, and run gel at 200 volts for approximately 40 minutes.
Image capture using the GelDoc system.

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Description

Delve into the fascinating world of SDS-PAGE electrophoresis and polyacrylamide gels. This quiz explores the principles of protein separation, the chemistry behind gel formation, and the properties of amino acids. Test your knowledge on the methods and implications of these techniques in biochemistry.