Podcast
Questions and Answers
Why is it important to consider the composition of the extraction buffer before cell disruption?
Why is it important to consider the composition of the extraction buffer before cell disruption?
- To increase the solubility of insoluble materials.
- To maintain protein stability and activity during the extraction process. (correct)
- To minimize the need for protease inhibitors.
- To ensure complete cell lysis, regardless of the protein target.
You are purifying an intracellular protein that tends to aggregate. Which strategy would be LEAST effective in preventing aggregation during cell disruption?
You are purifying an intracellular protein that tends to aggregate. Which strategy would be LEAST effective in preventing aggregation during cell disruption?
- Using protease inhibitors.
- Removing insoluble material promptly after cell disruption.
- Maintaining a low ionic strength in the extraction buffer. (correct)
- Adding chelating agents like EDTA to the buffer.
What is the primary purpose of adding reducing agents, such as DTT, during cell disruption?
What is the primary purpose of adding reducing agents, such as DTT, during cell disruption?
- To increase the ionic strength of the extraction buffer.
- To promote proper protein folding and renaturation.
- To prevent the oxidation of proteins and maintain their reduced state. (correct)
- To inhibit protease activity by chelating metal ions.
Which of the following factors creates the MOST challenges when purifying extracellular proteins compared to intracellular proteins?
Which of the following factors creates the MOST challenges when purifying extracellular proteins compared to intracellular proteins?
What is the MOST likely reason for poor yield of a properly folded intracellular protein after purification from inclusion bodies?
What is the MOST likely reason for poor yield of a properly folded intracellular protein after purification from inclusion bodies?
Why is EDTA often used in bacterial cell lysis protocols, particularly for Gram-positive bacteria?
Why is EDTA often used in bacterial cell lysis protocols, particularly for Gram-positive bacteria?
Which cell disruption method is LEAST suitable for processing large sample volumes due to the risk of local overheating?
Which cell disruption method is LEAST suitable for processing large sample volumes due to the risk of local overheating?
When disrupting plant cells with fibrous tissue, why is it recommended to grind the sample in liquid nitrogen?
When disrupting plant cells with fibrous tissue, why is it recommended to grind the sample in liquid nitrogen?
Why is it important to add protease inhibitors during cell disruption of plant and eukaryotic cells?
Why is it important to add protease inhibitors during cell disruption of plant and eukaryotic cells?
How does a French press disrupt cells?
How does a French press disrupt cells?
For which type of cells is disruption by osmosis with hypotonic buffer most appropriate?
For which type of cells is disruption by osmosis with hypotonic buffer most appropriate?
What principle is used in precipitation methods to fractionate complex protein mixtures?
What principle is used in precipitation methods to fractionate complex protein mixtures?
Autolysis is a cell disruption method sometimes used for yeast. What substance is typically added to facilitate this process, and for approximately how long is the process allowed to occur?
Autolysis is a cell disruption method sometimes used for yeast. What substance is typically added to facilitate this process, and for approximately how long is the process allowed to occur?
What is the fundamental principle behind liquid chromatography?
What is the fundamental principle behind liquid chromatography?
According to the Hofmeister series, which type of salts are generally considered good and gentle precipitants for protein precipitation?
According to the Hofmeister series, which type of salts are generally considered good and gentle precipitants for protein precipitation?
What is the primary purpose of using ultrafiltration in protein purification?
What is the primary purpose of using ultrafiltration in protein purification?
In fractionated ammonium sulfate precipitation, what principle allows for the separation of different proteins?
In fractionated ammonium sulfate precipitation, what principle allows for the separation of different proteins?
What is the main function of dialysis in protein purification?
What is the main function of dialysis in protein purification?
Which of the following factors is primarily responsible for the separation of molecules in size exclusion chromatography?
Which of the following factors is primarily responsible for the separation of molecules in size exclusion chromatography?
Which characteristic of ultrafiltration membranes is most important for determining which proteins will be retained?
Which characteristic of ultrafiltration membranes is most important for determining which proteins will be retained?
In the context of liquid chromatography, what distinguishes FPLC from standard LC?
In the context of liquid chromatography, what distinguishes FPLC from standard LC?
Which of the following methods separates proteins based on their specific binding affinity for a particular molecule?
Which of the following methods separates proteins based on their specific binding affinity for a particular molecule?
What is the role of pressure, vacuum, or centrifugation in ultrafiltration?
What is the role of pressure, vacuum, or centrifugation in ultrafiltration?
The Biuret assay relies on a color reaction. Which substance and protein functional group are critical to this reaction?
The Biuret assay relies on a color reaction. Which substance and protein functional group are critical to this reaction?
What is a key limitation of the Biuret assay that researchers must consider when selecting a protein quantification method?
What is a key limitation of the Biuret assay that researchers must consider when selecting a protein quantification method?
Which advantage makes the BCA assay particularly useful in complex experimental setups?
Which advantage makes the BCA assay particularly useful in complex experimental setups?
A researcher needs to quantify a protein sample but knows it contains reducing agents. Which assay should they avoid?
A researcher needs to quantify a protein sample but knows it contains reducing agents. Which assay should they avoid?
The Bradford assay is known for its speed and sensitivity, but what factor introduces a degree of subjectivity to its measurements?
The Bradford assay is known for its speed and sensitivity, but what factor introduces a degree of subjectivity to its measurements?
For quantifying proteins using UV absorption, why is a differential measurement at 235 nm and 280 nm preferred over just measuring at 280 nm?
For quantifying proteins using UV absorption, why is a differential measurement at 235 nm and 280 nm preferred over just measuring at 280 nm?
Why is measuring absorption at 205 nm considered a very objective method for protein quantification, despite not being the wavelength of maximum absorption for peptide bonds?
Why is measuring absorption at 205 nm considered a very objective method for protein quantification, despite not being the wavelength of maximum absorption for peptide bonds?
What makes the fluorescence assay with OPA the most sensitive method for protein quantification, and what is a significant drawback of this method?
What makes the fluorescence assay with OPA the most sensitive method for protein quantification, and what is a significant drawback of this method?
A protein sample contains a metal center that absorbs visible light. Which protein quantification method would be most appropriate for this sample?
A protein sample contains a metal center that absorbs visible light. Which protein quantification method would be most appropriate for this sample?
Before beginning protein purification, what is a crucial initial consideration regarding the starting material?
Before beginning protein purification, what is a crucial initial consideration regarding the starting material?
In discontinuous electrophoresis, what is the primary purpose of the stacking gel?
In discontinuous electrophoresis, what is the primary purpose of the stacking gel?
What is the fundamental principle behind Isoelectric Focusing (IEF)?
What is the fundamental principle behind Isoelectric Focusing (IEF)?
How are Immobilines used in isoelectric focusing (IEF) to establish a stable pH gradient?
How are Immobilines used in isoelectric focusing (IEF) to establish a stable pH gradient?
In two-dimensional gel electrophoresis (2D-GE), what property is used to separate proteins in the first dimension?
In two-dimensional gel electrophoresis (2D-GE), what property is used to separate proteins in the first dimension?
What is the primary advantage of capillary electrophoresis (CE) compared to traditional gel electrophoresis?
What is the primary advantage of capillary electrophoresis (CE) compared to traditional gel electrophoresis?
What is electroendosmotic flow in capillary electrophoresis and how does it impact the separation?
What is electroendosmotic flow in capillary electrophoresis and how does it impact the separation?
In Western blotting, what is the purpose of transferring proteins from the gel to a membrane?
In Western blotting, what is the purpose of transferring proteins from the gel to a membrane?
After the transfer step in Western blotting, the membrane is typically blocked. What is the purpose of this blocking step?
After the transfer step in Western blotting, the membrane is typically blocked. What is the purpose of this blocking step?
In ion exchange chromatography (IEX), what property of proteins is primarily exploited for separation?
In ion exchange chromatography (IEX), what property of proteins is primarily exploited for separation?
Which type of interaction is used in hydrophobic interaction chromatography (HIC)?
Which type of interaction is used in hydrophobic interaction chromatography (HIC)?
In size exclusion chromatography (SEC), also known as gel filtration, what property of molecules determines their elution order?
In size exclusion chromatography (SEC), also known as gel filtration, what property of molecules determines their elution order?
What is the primary principle behind affinity chromatography?
What is the primary principle behind affinity chromatography?
What potential issue can arise from a strong interaction between a target protein and a ligand in affinity chromatography?
What potential issue can arise from a strong interaction between a target protein and a ligand in affinity chromatography?
Which of the following elutions is LEAST likely to disrupt protein structure?
Which of the following elutions is LEAST likely to disrupt protein structure?
In immobilized metal ion affinity chromatography (IMAC), which amino acid residues commonly participate in metal ion complex formation?
In immobilized metal ion affinity chromatography (IMAC), which amino acid residues commonly participate in metal ion complex formation?
A researcher wants to purify a recombinant protein with a His6 tag using IMAC. After binding the protein to the column, what elution strategy would be MOST appropriate?
A researcher wants to purify a recombinant protein with a His6 tag using IMAC. After binding the protein to the column, what elution strategy would be MOST appropriate?
What is the MAIN principle underlying separation in electrophoresis?
What is the MAIN principle underlying separation in electrophoresis?
How does the viscosity of the solution affect electrophoretic mobility (µ)?
How does the viscosity of the solution affect electrophoretic mobility (µ)?
What determines the pore size in an agarose gel used for electrophoresis?
What determines the pore size in an agarose gel used for electrophoresis?
What are the two key components that make up a polyacrylamide gel?
What are the two key components that make up a polyacrylamide gel?
In polyacrylamide gel electrophoresis, what do the terms 'T' and 'C' represent?
In polyacrylamide gel electrophoresis, what do the terms 'T' and 'C' represent?
Which staining method is generally MORE sensitive for detecting proteins in a gel?
Which staining method is generally MORE sensitive for detecting proteins in a gel?
Ethidium bromide is primarily used to visualize which type of molecule in electrophoresis?
Ethidium bromide is primarily used to visualize which type of molecule in electrophoresis?
Flashcards
Biuret Assay
Biuret Assay
A method for protein quantification based on a color reaction with biuret and copper sulfate.
BCA Assay
BCA Assay
A protein quantification method combining Biuret assay with Bicinchoninic acid (BCA).
Bradford Assay
Bradford Assay
A quick protein quantification method using Coomassie Brilliant Blue G 250 dye that binds to certain amino acids.
Absorption at 280 nm
Absorption at 280 nm
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Absorption at 205 nm
Absorption at 205 nm
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Fluorescence Assay
Fluorescence Assay
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Chromophores
Chromophores
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Sensitivity of BCA
Sensitivity of BCA
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Subjectivity of Bradford Assay
Subjectivity of Bradford Assay
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Starting Material Considerations
Starting Material Considerations
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Hofmeister series
Hofmeister series
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Chaotropic salts
Chaotropic salts
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Cosmotropic salts
Cosmotropic salts
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Fractionated ammonium sulfate precipitation
Fractionated ammonium sulfate precipitation
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Ultrafiltration
Ultrafiltration
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Molecular weight cut-off
Molecular weight cut-off
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Dialysis
Dialysis
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Liquid chromatography (LC)
Liquid chromatography (LC)
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Gas chromatography (GC)
Gas chromatography (GC)
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Fast Protein Liquid Chromatography (FPLC)
Fast Protein Liquid Chromatography (FPLC)
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Intracellular proteins
Intracellular proteins
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Extracellular proteins
Extracellular proteins
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Protease inhibitors
Protease inhibitors
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Reducing agents
Reducing agents
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Chelating agents
Chelating agents
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Nucleic acids and lipoproteins removal
Nucleic acids and lipoproteins removal
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EDTA-Ca-complex
EDTA-Ca-complex
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Enzymatic cell disruption
Enzymatic cell disruption
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French press
French press
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Ultrasonic disintegration
Ultrasonic disintegration
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Cell mill with glass beads
Cell mill with glass beads
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Cold homogenization buffer
Cold homogenization buffer
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SYPRO Dyes
SYPRO Dyes
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SDS Electrophoresis
SDS Electrophoresis
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Isoelectric Focusing (IEF)
Isoelectric Focusing (IEF)
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Two-Dimensional Electrophoresis (2D)
Two-Dimensional Electrophoresis (2D)
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Capillary Electrophoresis (CE)
Capillary Electrophoresis (CE)
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Electroendosmotic Flow
Electroendosmotic Flow
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Protein Molecular Weight (log Mr)
Protein Molecular Weight (log Mr)
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Discontinuous Electrophoresis
Discontinuous Electrophoresis
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HPLC
HPLC
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UPLC
UPLC
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Resolution (R)
Resolution (R)
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Efficiency in chromatography
Efficiency in chromatography
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Hydrophobic nature
Hydrophobic nature
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Cation exchange chromatography
Cation exchange chromatography
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Ion exchange chromatography
Ion exchange chromatography
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Hydrophobic interaction chromatography (HIC)
Hydrophobic interaction chromatography (HIC)
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Size exclusion chromatography (SEC)
Size exclusion chromatography (SEC)
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Affinity chromatography
Affinity chromatography
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IMAC
IMAC
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Ligand coupling
Ligand coupling
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Staining methods
Staining methods
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Electrophoretic mobility
Electrophoretic mobility
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Carrier matrices
Carrier matrices
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Study Notes
Enzyme Purification and Characterization
- This presentation covers enzyme purification and characterization, including protein properties, sample processing, chromatography, electrophoresis, enzyme activity, kinetics, inhibition, and industrial applications.
Content
- Introduction: Discusses chemical and physical properties of proteins.
- Protein content determination: Methods like Bradford, BCA, and UV are used.
- Cell disruption: Techniques for breaking open cells to release proteins.
- Sample concentration and clarification: Procedures to concentrate and purify the protein samples.
- Liquid chromatography: Different types (IAC, HIC, GAC, Affinity, IMAC) separate proteins based on properties.
- Electrophoresis: Techniques like SDS, IEF, 2-D-Elpho, and capillary elpho are mentioned, along with blotting.
- Enzymes: General properties and characteristics.
- Enzyme activity determination: Assays.
- Coupled enzyme activity assays: Assays involving coupled reactions.
- Enzyme kinetics: Examining the rates of enzyme-catalyzed reactions.
- Regulation of enzyme activity: Factors that control enzyme activity.
- Inhibition of enzymes: Mechanisms of enzyme inhibition.
- Industrial use of enzymes: Applications in various industries.
Literature
- Lottspeich and Engels (2018), Bioanalytics
- Bonner (2007), Protein purification
- Polaina and MacCabe (2007), Industrial Enzymes
- Rosenberg (1996), Protein analysis and purification
- Nelson and Cox (2008), Lehninger Principles of Biochemistry
- GE Healthcare (Cytiva) Strategies for protein purification Handbook
Protein Structure
- Discusses the essential step of protein purification—crucial for understanding its function.
- Includes details on DNA transcription into complementary RNA, RNA translation on Ribosome to polypeptide chain, polypeptide folding for the native structure, and the role of ATP and Glucose in the process.
Lab Course
- Covers topics, such as the purification of extracellular copper enzymes from fungi, chemo-enzymatic natural product synthesis (with purification of a flavoprotein), and gel electrophoresis and enzyme kinetics with a perhydrolase.
Proteins (Table 3-1)
- Provides a table of properties and conventions of amino acids in proteins, including abbreviations, molecular weights, pK values, pl, hydropathy index, and occurrence.
- Includes 20 common amino acids and details like glycine, alanine, proline, valine, leucine, isoleucine, methionine, etc.
- Covers unusual amino acid, selenocysteine.
Proteins (Graph/Diagram)
- Explains isoelectric point calculation for amino acids and its significance, focusing on titration and its connection to charge.
- Includes isoelectric pH values for various proteins.
Proteins (Secondary Structure)
- Defines protein secondary structures:
- α-helix: a right-handed helix formed by intramolecular hydrogen bonds.
- β -sheet: antiparallel or parallel arrangement with pleated sheets, showing hydrophobic tendency.
- β-turn: connecting secondary structure types, creating a 180-degree turn.
Proteins (Tertiary Structure)
- Describes tertiary structure formation from linked secondary structural elements.
- Includes small motifs (β-α-β loop) and prominent motifs (α/β barrel).
Proteins (Quaternary Structure)
- Defines quaternary structure as the arrangement of subunits forming a functional protein complex, often at key metabolic pathway control points.
- Illustrates this with hemoglobin.
Protein Folding Thermodynamics
- Illustrates the thermodynamics of protein folding as a funnel.
- Explains that the top of the funnel represents a wide range of protein conformations and high conformational entropy, while the bottom represents the native structure and reduced folding states.
Sickle-Cell Anemia
- Describes sickle-cell anemia as a result of a hemoglobin single point mutation (E → V).
- States that this leads to misfolded proteins and misfolded erythrocytes.
Creutzfeldt-Jakob Disease (Prion)
- Defines prions as "proteinaceous infectious particles."
- Explains that normal prion protein (PrPc) converts to a disease-causing form (PrPSc), leading to PrPSc accumulation and disease.
Conjugated Proteins (Table 3-4)
- Lists conjugated protein classes, including lipoproteins, glycoproteins, phosphoproteins, hemoproteins, and metalloproteins.
- Outlines examples, prosthetic groups, solubility, and molecular masses.
- Covers concepts such as biospecificity and post-translational modification.
Protein Purification
- Includes methods like ion exchange chromatography (IEX), native PAGE, hydrophobic interaction chromatography (HIC), reversed-phase chromatography, salting-in and salting-out, fractionations, and ammonium sulfate precipitation.
Protein Purification (Additional Information)
- Discusses size exclusion chromatography (SEC) for molecular mass analysis and ultrafiltration.
- Explains the importance of combining techniques and the potential loss of protein at each step.
- Provides a flow chart of a generic protein purification strategy.
- Uses a sample purification balance sheet to provide context.
Protein Quantification
- General principles: Explains colorimetric and spectrophotometric assays for protein assessment.
- Colorimetric Assays: Includes BIURET, BCA, and BRADFORD assays and their applications.
- Spectrophotometric Assays: Covers OPA-Fluorescence for protein quantification and measuring absorbance at 280 nm.
Absorption at 205 nm and 280 nm
- Analyzes absorbance at 205 nm (peptide bond), 280 nm (aromatic amino acids), 235 nm and 280nm for differential measurement.
Cell Disruption
- Describes the processes used to break apart cells, including cell wall-related considerations and various methods such as mechanical disruption, enzymatic techniques, and physical/chemical agents such as reducing agents and chelating agents.
- Additional details include the role of protease inhibitors, removal of insoluble material, isolation, purification, and the consideration of ionic strength.
- Explores different cell types (bacteria, yeast, plants, and higher eukaryotes), along with suitable disruption methods and important considerations.
- Explains ultrasonic disintegration and high-pressure homogenization in more detail as cell disruption methods.
Other Topics
- Ultrafiltration: Concentrates proteins using semipermeable membranes.
- Dialysis: Desalting and buffer exchange technique.
- Liquid Chromatography: Covers various chromatography types:
· Ion exchange (IEX)—Separating charged molecules.
· Hydrophobic interaction chromatography (HIC)—Separating proteins based on hydrophobicity.
· Size exclusion chromatography (SEC/Gel filtration)—Separating by size.
· Affinity chromatography—Using specific ligand-receptor interactions.
· Immobilized metal ion affinity chromatography (IMAC)—Using metal ions to bind specific proteins, especially those with histidine tags.
- Details associated with each type of chromatography including the principles of separation, practical considerations, and typical chromatograms.
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Description
Questions about cell lysis methods, buffer composition, and challenges in protein purification, including aggregation, yield, and differences between intracellular and extracellular proteins. Focuses on optimizing protein recovery and preventing degradation.
