Podcast
Questions and Answers
What is the primary purpose of using polyacrylamide gel electrophoresis (PAGE) in protein analysis?
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?
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?
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?
What property of amino acids allows proteins to act as both bases and acids, exhibiting amphoteric behavior?
How does the isoelectric point (pI) of a protein relate to its net electric charge?
How does the isoelectric point (pI) of a protein relate to its net electric charge?
In SDS-PAGE, what is the purpose of adding sodium dodecyl sulfate (SDS) to the protein samples?
In SDS-PAGE, what is the purpose of adding sodium dodecyl sulfate (SDS) to the protein samples?
Which of the following is NOT a benefit of using polyacrylamide gel electrophoresis for separating proteins?
Which of the following is NOT a benefit of using polyacrylamide gel electrophoresis for separating proteins?
What is the main reason why polyacrylamide gel electrophoresis is commonly used for separating DNA fragments?
What is the main reason why polyacrylamide gel electrophoresis is commonly used for separating DNA fragments?
What is the primary function of Sodium Dodecyl Sulfate (SDS) in SDS-PAGE?
What is the primary function of Sodium Dodecyl Sulfate (SDS) in SDS-PAGE?
Why is the amount of SDS bound to a protein proportional to its size?
Why is the amount of SDS bound to a protein proportional to its size?
What is the purpose of using reducing agents like beta-mercaptoethanol or DTT in SDS-PAGE?
What is the purpose of using reducing agents like beta-mercaptoethanol or DTT in SDS-PAGE?
How does SDS-PAGE contribute to the accurate determination of protein molecular weights?
How does SDS-PAGE contribute to the accurate determination of protein molecular weights?
Which of the following is NOT a benefit of using SDS in SDS-PAGE?
Which of the following is NOT a benefit of using SDS in SDS-PAGE?
What is the primary reason for denaturing proteins using SDS in SDS-PAGE?
What is the primary reason for denaturing proteins using SDS in SDS-PAGE?
Why is it important to sterilize, filter, and perform quality control on Fetal Bovine Serum (FBS) used in cell cultures?
Why is it important to sterilize, filter, and perform quality control on Fetal Bovine Serum (FBS) used in cell cultures?
What is the primary purpose of conducting SDS-PAGE? Select the most comprehensive answer.
What is the primary purpose of conducting SDS-PAGE? Select the most comprehensive answer.
Which of the following statements accurately describes the migration of proteins in SDS-PAGE?
Which of the following statements accurately describes the migration of proteins in SDS-PAGE?
What is the role of the polyacrylamide gel in SDS-PAGE?
What is the role of the polyacrylamide gel in SDS-PAGE?
What is the correct dilution ratio for the 10x electrophoresis buffer with double-distilled water?
What is the correct dilution ratio for the 10x electrophoresis buffer with double-distilled water?
Which component is mixed with the Laemmli buffer containing beta-mercaptoethanol for sample preparation?
Which component is mixed with the Laemmli buffer containing beta-mercaptoethanol for sample preparation?
What is the thermal denaturation temperature and duration for the prepared samples?
What is the thermal denaturation temperature and duration for the prepared samples?
In preparing sample number 2 (FBS), what is the volume of water used?
In preparing sample number 2 (FBS), what is the volume of water used?
Which of the following is NOT listed as a reagent for the SDS-PAGE procedure?
Which of the following is NOT listed as a reagent for the SDS-PAGE procedure?
What is the primary function of BSA in biochemical protocols?
What is the primary function of BSA in biochemical protocols?
Which of the following proteins is NOT typically found in milk?
Which of the following proteins is NOT typically found in milk?
What characteristic of BSA makes it suitable for use in molecular biology?
What characteristic of BSA makes it suitable for use in molecular biology?
In terms of molecular weight, how does casein compare to other milk proteins?
In terms of molecular weight, how does casein compare to other milk proteins?
Which of the following functions is NOT associated with BSA in laboratory settings?
Which of the following functions is NOT associated with BSA in laboratory settings?
What is the mass range of casein found in milk?
What is the mass range of casein found in milk?
Which component is NOT one of the main groups found in milk?
Which component is NOT one of the main groups found in milk?
What role does BSA play in relation to oxidizing agents during enzyme storage?
What role does BSA play in relation to oxidizing agents during enzyme storage?
Which of the following is a primary protein source in human nutrition derived from milk?
Which of the following is a primary protein source in human nutrition derived from milk?
Which of these statements about immunoglobulins in milk is true?
Which of these statements about immunoglobulins in milk is true?
What is the primary factor that influences the migration rate of proteins during electrophoresis?
What is the primary factor that influences the migration rate of proteins during electrophoresis?
Which agent is commonly used to initiate the polymerization of acrylamide gels?
Which agent is commonly used to initiate the polymerization of acrylamide gels?
How does the cross-linking density of acrylamide gels affect protein migration?
How does the cross-linking density of acrylamide gels affect protein migration?
What is the primary purpose of sodium dodecyl sulfate (SDS) in protein electrophoresis?
What is the primary purpose of sodium dodecyl sulfate (SDS) in protein electrophoresis?
What characteristic of proteins can affect their migration through polyacrylamide gel besides mass?
What characteristic of proteins can affect their migration through polyacrylamide gel besides mass?
Which variable is NOT directly adjustable in the preparation of polyacrylamide gels?
Which variable is NOT directly adjustable in the preparation of polyacrylamide gels?
What may pose a serious health hazard during the use of polyacrylamide gels?
What may pose a serious health hazard during the use of polyacrylamide gels?
The migration rate of polypeptides in gel electrophoresis can be primarily influenced by their:
The migration rate of polypeptides in gel electrophoresis can be primarily influenced by their:
What occurs to proteins when treated with SDS?
What occurs to proteins when treated with SDS?
In protein electrophoresis, why are proteins with a higher molecular weight more likely to migrate slower?
In protein electrophoresis, why are proteins with a higher molecular weight more likely to migrate slower?
Flashcards
SDS-PAGE
SDS-PAGE
A method for separating proteins using an electric field in polyacrylamide gel.
Isoelectric point (pI)
Isoelectric point (pI)
The pH at which a protein has no net electric charge.
Polyacrylamide gel
Polyacrylamide gel
The medium used in electrophoresis that separates proteins and nucleotides.
Amphoteric properties
Amphoteric properties
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Acrylamide
Acrylamide
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Protein separation range
Protein separation range
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Cross-linking agent
Cross-linking agent
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Free-radical polymerization
Free-radical polymerization
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SDS
SDS
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Hydrophobic groups
Hydrophobic groups
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Negative charge in proteins
Negative charge in proteins
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Migration towards anode
Migration towards anode
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Molecular weight determination
Molecular weight determination
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Protein denaturation
Protein denaturation
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Reducing agents
Reducing agents
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Electrophoresis buffer
Electrophoresis buffer
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Fetal Bovine Serum (FBS)
Fetal Bovine Serum (FBS)
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Homogeneity of proteins
Homogeneity of proteins
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Laemmli buffer
Laemmli buffer
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Thermal denaturation
Thermal denaturation
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Molecular weight markers
Molecular weight markers
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BSA
BSA
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Uses of BSA
Uses of BSA
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Blocking agent
Blocking agent
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Western blotting
Western blotting
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Nitrocellulose
Nitrocellulose
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Casein
Casein
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Alpha-lactoalbumin
Alpha-lactoalbumin
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Beta-lactoglobulin
Beta-lactoglobulin
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Immunoglobulins
Immunoglobulins
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Cell culture nutrient
Cell culture nutrient
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Electric field strength
Electric field strength
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Protein electrophoresis
Protein electrophoresis
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N,N-methylenebisacrylamide
N,N-methylenebisacrylamide
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Polymerization
Polymerization
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Migration rate
Migration rate
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Protein-SDS complex
Protein-SDS complex
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Denaturing conditions
Denaturing conditions
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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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