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?

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

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

<p>Investigating the interaction between proteins. (D)</p> Signup and view all the answers

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

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

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

<p>SDS disrupts the protein structure, creating more binding sites for the stain. (C)</p> 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?

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

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

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

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

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

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

<p>40 µl (A)</p> Signup and view all the answers

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

<p>Well 5 (A)</p> Signup and view all the answers

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

<p>2 µl (D)</p> Signup and view all the answers

What is the volume of buffer used in well 7?

<p>10 µl (B)</p> 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?

<p>It is the least among all wells. (C)</p> Signup and view all the answers

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

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

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

<p>N,N-methylenebisacrylamide (A)</p> Signup and view all the answers

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

<p>The pH of the solution (A)</p> Signup and view all the answers

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

<p>They gain additional electric charge (A)</p> Signup and view all the answers

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

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

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

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

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

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

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

<p>It is a potent neurotoxin (C)</p> Signup and view all the answers

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

<p>To mask the original charges of proteins (A)</p> Signup and view all the answers

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

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

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

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

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

<p>Differences in protein mass (C)</p> Signup and view all the answers

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

<p>The presence of reducing agents (B)</p> Signup and view all the answers

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

<p>It promotes the polymerization reaction of acrylamide (A)</p> Signup and view all the answers

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

<p>It influences the migration rate of proteins (B)</p> Signup and view all the answers

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

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

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

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

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

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

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

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

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

<p>It stabilizes the enzyme functions (D)</p> Signup and view all the answers

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

<p>BSA (A)</p> Signup and view all the answers

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

<p>10 to 250 Da (D)</p> Signup and view all the answers

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

<p>Dilute with 800 ml of ddH2O (B)</p> Signup and view all the answers

Which group of proteins is primarily found in milk?

<p>Caseins (A)</p> Signup and view all the answers

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

<p>To reduce disulfide bonds (A)</p> Signup and view all the answers

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

<p>Bovine serum albumin (BSA) (A)</p> Signup and view all the answers

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

<p>Lipids (A), Sugars (C)</p> Signup and view all the answers

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

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

Flashcards

Protein Electrophoresis

A method for separating molecules by electric charge.

SDS-PAGE

A type of gel electrophoresis used to separate proteins based on size.

Polyacrylamide Gel

A gel used as an electrophoretic medium for separating proteins.

Isoelectric Point (pI)

The pH at which a protein carries no net electric charge.

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Amphoteric Properties

Properties of amino acids that allow them to act as acids or bases.

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Cross-Linking Agent

A compound that links polymer chains in gel preparation.

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Electric Field Strength

Intensity of the electric field affecting ion movement in electrophoresis.

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Staining Methods

Techniques used to visualize separated DNA fractions in gels.

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Acrylamide Monomers

Small chemical units that polymerize to form polyacrylamide gel.

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N,N-methylenebisacrylamide

A cross-linking agent that helps form polyacrylamide gels.

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Ammonium Persulfate

A chemical initiator for the polymerization of acrylamide to form gels.

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Sodium Dodecyl Sulfate

An ionic detergent used to give proteins a consistent negative charge.

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Migration Rate

The speed at which proteins move through the gel during electrophoresis.

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Cross-Linking Density

The amount of cross-linking in the gel, influencing protein separation.

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Denaturing Conditions

Conditions that disrupt protein structure for uniform separation by mass.

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Sample Preparation

Combining specific amounts of substances to create a total volume for experimentation.

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Gel Sample Volume

Total volume of samples in gel electrophoresis is typically 40 µl per well.

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FBS Composition

Fetal Bovine Serum (FBS) is prepared using 29 µl Water, 1.5 µl Protein, and 10 µl Buffer.

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BSA Concentration

Bovine Serum Albumin (BSA) is used at 2 µl concentration in the sample preparation.

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PCR Mix Ingredients

The PCR mix includes 25 µl Water, 5 µl Protein, and 10 µl Buffer for preparation.

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BSA Uses

BSA is used to block surfaces in assays, stabilize enzymes, and as a nutrient.

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Western Blot

A technique for detecting specific proteins using labeled antibodies.

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ELISA

An assay to detect and quantify proteins or hormones via antigen-antibody interactions.

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Laemmli Buffer

A buffer used to prepare samples for SDS-PAGE by denaturing proteins.

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Fetal Bovine Serum (FBS)

A nutrient-rich serum used in cell culture to support growth.

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Nitrocellulose Blocking

Using BSA to block binding sites on nitrocellulose membranes in assays.

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Protein Molecular Weight Marker

Standards with known sizes used to estimate unknown protein sizes in gels.

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Casein Protein

The main protein in milk important for nutrition and food products.

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Dilution in SDS-PAGE

Mixing concentrated solutions with water to prepare samples for gel electrophoresis.

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SDS

Sodium Dodecyl Sulfate, a detergent used to denature proteins.

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Denaturation

The process of altering a protein's structure, causing it to unfold.

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Covalent Bonds

Strong chemical bonds involving shared electron pairs, like disulfide bridges in proteins.

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Reducing Agents

Chemicals that break disulfide bonds in proteins; includes beta-mercaptoethanol and DTT.

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Molecular Weight Determination

The process of determining the size of proteins based on their movement in a gel.

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Protein Mixture Identification

Analyzing the composition and types of proteins in a sample using electrophoresis.

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Homogeneity Check

Assessing whether a protein sample consists of a single type of protein or multiple types.

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BSA

Bovine Serum Albumin, a common protein used in research and biochemistry.

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FBS

Fetal Bovine Serum used in cell cultures, derived from calf blood.

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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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Exercise no. 9: Protein Electrophoresis via SDS-PAGE Method PDF

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