Intracellular Protein Purification
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Questions and Answers

When purifying an intracellular protein, what is the first major step after cell disruption, and why is this step crucial for downstream purification processes?

Removal of insoluble material. This is crucial because insoluble material can interfere with downstream purification techniques, potentially clogging columns or reducing the effectiveness of subsequent steps.

In the context of protein purification, what challenges are specifically associated with the renaturation of proteins isolated from inclusion bodies, and what buffer conditions are typically used?

Proteins can be difficult to refold correctly, leading to aggregation and low yields. Low ionic strength buffers are typically used to solubilize the protein, but optimized conditions are protein specific.

When preparing a cell disruption protocol for intracellular protein purification, why are protease inhibitors added to the extraction buffer? Name one.

Protease inhibitors are added to prevent the degradation of the target protein by cellular proteases released during cell lysis. An example would be EDTA.

Explain the principle behind using reducing agents like DTT in cell disruption buffers, particularly in the context of protein stability and activity.

<p>Reducing agents like DTT maintain a reduced environment, preventing the formation of unwanted disulfide bonds in proteins. They are typically used to maintain protein stability and activity.</p> Signup and view all the answers

Besides protease inhibitors, reducing agents and chelating agents, name three other factors to consider when preparing cell disruption protocols?

<p>Extraction buffer composition, addition of enzyme substrates / inhibitors / activators / cofactors.</p> Signup and view all the answers

During protein purification, why is it crucial to minimize the number of purification steps?

<p>To minimize the loss of the target protein at each step, which can significantly reduce the overall yield.</p> Signup and view all the answers

List three factors that contribute to protein loss or degradation during a purification procedure.

<ol> <li>Proteolytic activity 2. Temperature and pH instability 3. Interfaces 4. Divalent metal ions</li> </ol> Signup and view all the answers

Why is it important to perform empirical determination of a procedure's efficacy at every stage in a protein purification strategy?

<p>To ensure that each technique produces the desired increase in yield or specific activity and to identify ineffective steps that should be replaced.</p> Signup and view all the answers

Explain how the choice of starting material (tissue or cell type) can impact a protein purification strategy.

<p>Different tissues or cells will have different levels of expression of the target protein, as well as different types and amounts of contaminating proteins, affecting the required purification steps.</p> Signup and view all the answers

Describe the role of 'pre-cleaning by precipitation' in a protein purification flow chart and name a reagent commonly used for this purpose.

<p>Pre-cleaning removes bulk contaminants, reducing the load on subsequent chromatography steps. Ammonium sulfate</p> Signup and view all the answers

Explain how you would use SDS-PAGE in the context of protein purification.

<p>To observe the purity of the protein at various stages of purification.</p> Signup and view all the answers

Differentiate between Ion Exchange Chromatography (IEX) and Affinity Chromatography in terms of the types of proteins they are typically used to purify and their underlying principles.

<p>IEX (Ion Exchange Chromatography) is used for native proteins and separates based on charge. Affinity Chromatography is used for recombinant proteins and separates by binding affinity.</p> Signup and view all the answers

A protein sample has a volume of 20 ml and a protein concentration of 5 mg/ml. The activity assay shows 100 U/ml. Calculate the Total protein content, Total activity, and Specific Activity. Express answers with units.

<p>Total protein content (TP) = 100 mg. $TP = 5 \frac{mg}{ml} * 20 ml = 100 mg$. Total activity (TA) = 2000 U. $TA = 100 \frac{U}{ml} * 20 ml = 2000 U$. Specific Activity = 20 U/mg. $SA = \frac{2000 U}{100 mg} = 20 \frac{U}{mg}$</p> Signup and view all the answers

Why is it important to remove nucleic acids and lipoproteins during cell lysis, and what impact would their presence have on downstream processes?

<p>Nucleic acids and lipoproteins increase sample viscosity, which can interfere with downstream processes such as chromatography or protein purification. Removing them improves the efficiency and resolution of these processes.</p> Signup and view all the answers

How does EDTA aid in cell disruption, particularly for Gram-positive bacteria?

<p>EDTA chelates calcium ions, which are important for maintaining the integrity of the peptidoglycan cell wall in Gram-positive bacteria. By removing calcium, EDTA weakens the cell wall and renders it more permeable, facilitating cell lysis.</p> Signup and view all the answers

Compare and contrast the cell disruption methods suitable for Gram-positive versus Gram-negative bacteria, highlighting the differences in their cell wall structures.

<p>Gram-positive bacteria can be disrupted using enzymatic methods with lysozyme or EDTA/Tris due to their peptidoglycan cell wall. Gram-negative bacteria, with their more complex outer membrane, often require mechanical disruption like a cell mill with glass beads or freeze-thaw cycles.</p> Signup and view all the answers

Why is the freeze-thaw method not recommended for processing large volumes of cells?

<p>The freeze-thaw method is not suitable for large amounts because of local overheating, which can denature proteins or degrade other cellular components, reducing the yield and quality of the desired products.</p> Signup and view all the answers

What modifications are necessary when using a Waring blender to disrupt fibrous plant tissue, and why?

<p>When using a Waring blender to disrupt fibrous plant tissue, it's important to add DTT (dithiothreitol) and PVP (polyvinylpyrrolidone) to the buffer. DTT helps to reduce disulfide bonds, which can stabilize proteins and make the tissue more resistant to disruption. PVP binds to polyphenols released during disruption, preventing them from binding to and inactivating proteins.</p> Signup and view all the answers

Why are protease inhibitors often added during the disruption of higher eukaryotic cells, especially those from suspension culture?

<p>Protease inhibitors are added to prevent the degradation of proteins by endogenous proteases released during cell lysis. Higher eukaryotic cells, especially those from suspension culture, are very damageable and prone to protease release, so protease inhibitors are crucial for preserving protein integrity.</p> Signup and view all the answers

Explain how ultrasonic disintegration disrupts cells and what factors might affect its efficiency?

<p>Ultrasonic disintegration disrupts cells by generating intense sonic pressure waves in liquid suspensions, causing cavitation and cell lysis. Factors affecting efficiency include the amplitude and duration of sonication, sample viscosity, and temperature, as overheating can occur.</p> Signup and view all the answers

How does a high-pressure homogenizer (French press) disrupt cells, and why might multiple passes be necessary for certain cell types like yeast?

<p>A high-pressure homogenizer disrupts cells by forcing them through a small aperture at high pressure, causing shear forces that break the cell membrane. Multiple passes may be necessary for cells like yeast due to their robust cell walls, making them more resistant to disruption.</p> Signup and view all the answers

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

<p>At its isoelectric point, a protein has no net overall charge.</p> Signup and view all the answers

Describe the role of hydrogen bonds in the formation of the alpha-helix secondary structure in proteins.

<p>Intra-chain hydrogen bonds between the rigid peptide bonds stabilize the right-handed helix.</p> Signup and view all the answers

Explain how the amino acid composition of a segment of a protein influences its likelihood of forming a beta-sheet.

<p>Amino acids that are more hydrophobic and have a higher propensity for beta-sheets will favor the formation of this structure.</p> Signup and view all the answers

What is the main difference between tertiary and quaternary protein structure?

<p>Tertiary structure refers to the overall 3D arrangement of a single polypeptide chain, while quaternary structure involves the arrangement of multiple polypeptide chains (subunits) in a multi-subunit protein.</p> Signup and view all the answers

Describe the 'protein folding funnel' concept in thermodynamics.

<p>The protein folding funnel illustrates how a large number of possible unfolded protein conformations are gradually reduced to a single, stable native conformation through folding intermediates, guided by decreasing conformational entropy.</p> Signup and view all the answers

Explain how a single point mutation in hemoglobin, such as E -> V in sickle-cell anemia, can lead to misfolding and disease.

<p>The mutation alters the amino acid sequence, changing the protein's structure and causing it to misfold, which leads to the aggregation of misfolded proteins and misshapen erythrocytes.</p> Signup and view all the answers

Briefly describe prions, and how they cause disease.

<p>Prions are infectious proteins that can induce normally folded proteins to misfold into the prion form, leading to diseases like Creutzfeldt-Jakob disease.</p> Signup and view all the answers

How can manipulating the ionic strength of a solution affect protein solubility, and what are the terms for these effects?

<p>Increasing ionic strength can initially increase protein solubility ('salting in'), but at high concentrations, it can decrease solubility, causing precipitation ('salting out').</p> Signup and view all the answers

What properties of a protein are exploited in hydrophobic interaction chromatography?

<p>The hydrophobic nature of the protein is exploited, specifically hydrophobic amino acids on the protein surface.</p> Signup and view all the answers

When would you use ion exchange chromatography (IEX) for protein purification, and what property of the protein is key to this method?

<p>IEX is used when proteins have differing surface charges at a particular pH. The key property is the protein's net charge at the chosen pH.</p> Signup and view all the answers

Describe the characteristics and purpose of a beta-turn.

<p>Beta-turns are 180 degree turns involving four amino acid residues connecting secondary structure types.</p> Signup and view all the answers

How does knowledge of a protein's pI assist in choosing appropriate conditions for ion exchange chromatography?

<p>The pI helps in selecting the appropriate buffer pH and type of ion exchange resin (anion or cation exchanger) to ensure the protein binds to the column.</p> Signup and view all the answers

What is the role of 'salting out' in protein purification, and why does it occur at high salt concentrations?

<p>Salting out is used to precipitate proteins from solution. At high salt concentrations, salt ions compete with proteins for water molecules, reducing the protein's solubility and causing it to precipitate.</p> Signup and view all the answers

Explain how reversed-phase chromatography separates proteins.

<p>Reversed-phase chromatography separates proteins based on their hydrophobicity, using a hydrophobic stationary phase and an increasingly hydrophobic mobile phase to elute proteins in order of increasing hydrophobicity.</p> Signup and view all the answers

How do post-translational modifications influence protein bio-specificity?

<p>Post-translational modifications affect bio-specificity by altering a protein's ability to interact with other molecules</p> Signup and view all the answers

Explain how the Hofmeister series can be used to predict the effectiveness of different salts in protein precipitation.

<p>The Hofmeister series ranks ions by their ability to salt-out or salt-in proteins. Antichaotropic salts (cosmotropic) promote hydrophobic interactions and are good precipitants, while chaotropic salts disrupt these interactions and are not useful for precipitation.</p> Signup and view all the answers

Describe the steps involved in fractionated ammonium sulfate precipitation and why it is useful in protein purification.

<p>Fractionated ammonium sulfate precipitation involves adjusting the ammonium sulfate concentration in steps, precipitating different proteins at different saturation levels. After each step, the precipitate is separated (e.g., by centrifugation). This allows for selective enrichment of target proteins.</p> Signup and view all the answers

What is ultrafiltration and what are the different methods to perform it?

<p>Ultrafiltration concentrates proteins by filtration through a semipermeable membrane. Pressure, vacuum, or centrifugation are used to drive the solution through the membrane, retaining larger molecules.</p> Signup and view all the answers

Describe the principle behind dialysis and its common application in protein purification.

<p>Dialysis is a technique for desalting or buffer exchange. It works by allowing small molecules to diffuse across a semipermeable membrane while retaining larger proteins. This separates proteins from unwanted salts or changes the buffer composition.</p> Signup and view all the answers

How does the molecular weight cut-off (MWCO) of an ultrafiltration membrane affect the separation of proteins?

<p>The MWCO determines the size of molecules that can pass through the membrane. A membrane with a specific MWCO will retain proteins larger than that size while allowing smaller molecules to pass through, effectively separating proteins based on size.</p> Signup and view all the answers

Explain the fundamental difference between gas chromatography (GC) and liquid chromatography (LC).

<p>The main difference is the mobile phase; GC uses a gas as the mobile phase, whereas LC uses a liquid as the mobile phase.</p> Signup and view all the answers

Briefly describe the difference between column chromatography and surface chromatography.

<p>Column chromatography involves a solid stationary phase packed in a column. Surface chromatography does not use a column; instead, the stationary phase is spread over a surface.</p> Signup and view all the answers

List four different types of liquid chromatography based on their separation principle.

<p>Ion exchange, hydrophobic interaction, size exclusion, and affinity chromatography.</p> Signup and view all the answers

What is the key difference between LC and FPLC, and how does this difference affect their applications?

<p>FPLC (Fast Protein Liquid Chromatography) operates at higher pressures than standard LC. This allows for faster separation and higher resolution, making it suitable for purifying sensitive biomolecules like proteins.</p> Signup and view all the answers

Describe how you might use a combination of ammonium sulfate precipitation and size exclusion chromatography to purify a protein from a complex mixture.

<p>First, use ammonium sulfate precipitation to selectively precipitate a subset of proteins, including the target protein. Then, use size exclusion chromatography to separate proteins in the precipitate based on their size, further isolating the target protein from other proteins of different sizes.</p> Signup and view all the answers

Explain how resolution (R) is related to selectivity and efficiency in liquid chromatography.

<p>Resolution (R) reflects the ability to separate two peaks, which is determined by the selectivity (how well the column distinguishes between different molecules) and the efficiency (how narrow the peaks are).</p> Signup and view all the answers

Name four characteristics of molecules that can be exploited for separation using liquid chromatography.

<p>Hydrophobic nature, charge, size, and bio specificity.</p> Signup and view all the answers

In ion exchange chromatography (IEX), what is the key difference between cation exchange and anion exchange?

<p>Cation exchange chromatography uses a negatively charged resin to bind positively charged molecules, while anion exchange chromatography uses a positively charged resin to bind negatively charged molecules.</p> Signup and view all the answers

Describe the principle behind hydrophobic interaction chromatography (HIC).

<p>HIC separates proteins based on their hydrophobicity. Proteins with hydrophobic regions bind to a hydrophobic resin in high salt concentrations, and are eluted by decreasing the salt concentration.</p> Signup and view all the answers

How are proteins prepared for binding in hydrophobic interaction chromatography (HIC)?

<p>Proteins are typically prepared by dissolving them in a high-salt buffer to enhance hydrophobic interactions and promote binding to the HIC resin.</p> Signup and view all the answers

Explain the principle of size exclusion chromatography (SEC).

<p>SEC separates molecules based on their size. Smaller molecules enter the pores of the stationary phase (beads) and take a longer path through the column, while larger molecules cannot enter the pores and elute faster.</p> Signup and view all the answers

In size exclusion chromatography, how does particle size relate to elution volume?

<p>Larger particles elute earlier (smaller elution volume) because they cannot enter the pores of the stationary phase, whereas smaller particles elute later (larger elution volume) as they get trapped inside the pores.</p> Signup and view all the answers

Describe the general principle behind affinity chromatography.

<p>Affinity chromatography separates proteins based on a reversible, unique, bispecific interaction between the protein and an immobilized ligand. The protein binds specifically to the ligand, and is then eluted by disrupting this interaction.</p> Signup and view all the answers

Why are slow flow rates and long, thin columns preferred in affinity chromatography?

<p>Slow flow rates and long, thin columns promote interaction between the target protein and the immobilized ligand, increasing the chances of binding and improving the efficiency of the separation. This maximizes the opportunity for the protein to bind to the ligand.</p> Signup and view all the answers

Explain the principle of immobilized metal ion affinity chromatography (IMAC).

<p>IMAC separates proteins based on their affinity for metal ions (like nickel or cobalt) immobilized on a resin. Proteins with histidine (His) or cysteine residues, or those with engineered His-tags, bind to the metal ions. They are then eluted by competition, often using imidazole.</p> Signup and view all the answers

What is the purpose of using a His6 tag in recombinant proteins when using immobilized metal ion affinity chromatography (IMAC)?

<p>The His6 tag is a sequence of six histidine residues added to recombinant proteins to enhance their binding affinity to the metal ions on the IMAC resin, allowing for efficient purification.</p> Signup and view all the answers

Describe the principle of electrophoresis and how it separates molecules.

<p>Electrophoresis separates charged molecules based on their migration in an electric field. Separation is influenced by charge, size, and shape of the molecules, as well as the strength of the electric field and properties of the supporting matrix.</p> Signup and view all the answers

How does the pore size of an agarose gel affect the separation of molecules during electrophoresis?

<p>The pore size of agarose gel is determined by the concentration of agarose. Smaller pore sizes retard the movement of larger molecules, leading to better separation of molecules with different sizes. Larger pore sizes are used for separating large molecules.</p> Signup and view all the answers

How are polyacrylamide gels characterized, and what components are used in their formation?

<p>Polyacrylamide gels are characterized by T (total acrylamide concentration) and C (crosslinker concentration). They are formed from acrylamide monomers, a crosslinker (N, N-methylene bisacrylamide or Bis), and polymerization initiators.</p> Signup and view all the answers

Give two examples of staining methods used to visualize proteins in gels after electrophoresis.

<p>Coomassie Brilliant Blue staining and silver staining.</p> Signup and view all the answers

Flashcards

Intracellular proteins

Proteins located inside the cells, typically involved in cell function and processes.

Extracellular proteins

Proteins located outside the cells, often involved in communication between cells or structural support.

Cell disruption

The process of breaking open cells to extract proteins or other cellular components.

Protease inhibitors

Substances that prevent the activity of proteases, allowing the preservation of proteins during extraction.

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Reducing agents

Chemical substances that donate electrons to another substance, helping to maintain protein structure during purification.

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Fractionated Ammonium Sulfate Precipitation

A protein purification technique using ammonium sulfate to precipitate proteins by exploiting solubility differences.

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Size Exclusion Chromatography (SEC)

A method to separate proteins based on their size using a porous medium.

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Affinity Chromatography

A purification technique that uses specific binding interactions between proteins and ligands.

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Chromatography Steps

Sequential purification steps that may include IEX and affinity methods to enhance protein purity.

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Total Protein Content (TP)

Calculated as the product of protein concentration and volume in a sample.

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Total Activity (TA)

The measurement of enzymatic activity, reflecting the effectiveness of proteins in a sample.

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Specific Activity

A ratio that indicates the activity of a protein per unit of total protein content.

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Protein Purification Strategy

A systematic approach comprised of multiple techniques aimed at isolating a target protein.

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Lysozyme

An enzyme used to break down bacterial cell walls, specifically peptidoglycan.

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EDTA/Tris

Chemical agents used to increase the permeability of Gram-positive bacterial cell walls.

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Mechanical Disruption

Methods like cell mills or glass beads to physically break cells apart.

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Ultrasonic Disintegration

Breaks up cells using intense sonic pressure waves in liquid.

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High Pressure Homogenizers

Devices that shear cells as they pass through a small aperture to disrupt them.

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Autolysis

The self-digestion of cells, often used in yeast to break down cell structures.

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Precipitation in Protein Fractionation

Using differences in hydrophobic amino acids to separate proteins from mixtures.

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Hofmeister series

Classification of salts based on their effects on protein solubility.

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Kosmotropic salts

Salts that enhance hydrophobic interactions and promote protein precipitation.

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Chaotropic salts

Salts that disrupt hydrophobic interactions and prevent protein precipitation.

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(NH4)2SO4 precipitation

A method for fractionating proteins using varying percentages of saturation.

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Ultrafiltration

A technique that concentrates proteins by filtering through semipermeable membranes.

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Molecular weight cut-off

The specific size limit of molecules that can pass through a membrane in ultrafiltration.

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Dialysis

A technique used for desalting or changing buffers in protein samples.

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Liquid chromatography

A separation technique based on the distribution of compounds in two phases.

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Gas chromatography (GC)

A type of chromatography that uses gas as the mobile phase.

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FPLC

Fast Protein Liquid Chromatography, a variant of liquid chromatography for proteins.

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Protein purification

The process of isolating a specific protein from a mixture.

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Isoelectric point (pI)

The pH at which a protein has no net charge.

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Secondary structure

Local folded structures within a protein, such as α-helices and β-sheets.

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Tertiary structure

The overall 3D shape of a single protein molecule formed by various secondary structures.

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Quaternary structure

A complex of multiple protein subunits forming a larger functional unit.

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Hydrophobic interaction chromatography

A purification method based on the hydrophobic properties of proteins.

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Salting in

Increasing protein solubility at low salt concentrations.

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Salting out

Decreasing protein solubility at high salt concentrations.

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Sickle-cell anemia

A genetic disorder caused by a single mutation in hemoglobin that leads to misfolded proteins.

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Creutzfeldt-Jakob disease

A neurodegenerative disorder caused by misfolded proteins known as prions.

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Protein folding thermodynamics

The study of energy changes and conformations during protein folding.

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Hydrophobic amino acids

Amino acids with side chains that repel water, usually found in the protein core.

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Gel electrophoresis

A technique used to separate proteins based on size and charge.

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Protein bio specificity

The ability of a protein to interact with specific molecules or ligands.

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Amino acid composition

The unique sequence and arrangement of amino acids in a protein.

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Resolution (R)

The ability to distinguish between two peaks in chromatography.

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Efficiency

The performance of a chromatography method in terms of peak separation.

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Ion Exchange Chromatography (IEX)

A technique to separate ions and polar molecules based on their charge.

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Hydrophobic Interaction Chromatography (HIC)

A method for separating proteins based on their hydrophobic properties.

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Immobilized Metal Ion Affinity Chromatography (IMAC)

A technique using metal ions to capture proteins with specific residues.

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Agarose Gel

A gel made from agarose used for separating biomolecules by electrophoresis.

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Polyacrylamide Gel

A gel used for electrophoresis, created from acrylamide and a crosslinker.

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Electrophoretic Mobility

The speed at which a charged particle moves in an electric field, influenced by charge and size.

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Staining Methods

Techniques to visualize biomolecules in gels, such as Coomassie and silver staining.

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Protein Elution

The process of retrieving bound proteins from a chromatography matrix.

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Cation Exchange Chromatography

A specific type of ion exchange chromatography that swaps cations.

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Study Notes

Enzyme Purification and Characterization

  • This presentation covers enzyme purification and characterization
  • Basic protein properties are discussed (e.g., chemical and physical properties)
  • Determining protein content (Bradford, BCA, UV) is explained
  • Cell disruption methods are included (methods to break open cells to release proteins)
  • Sample concentration and clarification methods are detailed
  • Chromatography techniques (IAC, HIC, GAC, Affinity, IMAC) are described
  • Electrophoresis methods (SDS, IEF, 2-D-Elpho, capillary elpho) and blotting are discussed
  • Enzyme characteristics and properties are examined
  • Measuring enzyme activity (coupled enzyme activity assays) is explained
  • Enzyme kinetics and regulation are discussed
  • Enzymatic inhibition is analysed
  • Industrial applications of enzymes are covered

Literature

  • Relevant publications are referenced as:
    • F. Lottspeich, J. Engels (Eds.) Bioanalytics Wiley-VCH, 2018
    • P.L.R. Bonner Protein purification Taylor & Francis Group, 2007
    • J. Polaina, A.P. MacCabe Industrial Enzymes Springer Verlag, 2007
    • I.M. Rosenberg Protein analysis and purification Springer Verlag, 1996
    • D.L. Nelson, M.M. Cox Lehninger Principles of Biochemistry 4. Edition
    • GE Healthcare (Cytiva) Strategies for protein purification Handbook

Protein Structure

  • Proteins are made of a polypeptide chain folded into specific 3-D shapes
  • This 3D shape determines the function of the protein.
  • The folding into tertiary structure is governed by thermodynamics
  • The presentation explores the consequences of mutations and misfolding, including the cases of sickle-cell disease and Creutzfeld-Jakob disease.

Lab Course Topics

  • Extracellular copper fungal enzymes are mentioned
  • Chemo-enzymatic biotransformations are highlighted
  • Flavoprotein purification techniques are analysed
  • Gel electrophoresis and enzyme kinetics using perhydrolase are discussed

Amino Acids

  • A table of 20 amino acids is provided, including their abbreviations, chemical formulas, and physical properties
  • A formula for isoelectric point (pI) is referenced: E = 3.22 (H = 7.59)
  • Properties and conventions associated with amino acids are summarized
  • General structure of the L-amino acid components is explored
  • The importance of "unusual amino acids" is discussed

Isoelectric Point

  • Explanation of isoelectric point (pI) for amino acids and proteins
  • Describes isoelectric point determination in proteins
  • Shows how pI is characteristic of each amino acid

Protein Secondary Structures

  • The presentation covers alpha helices in proteins, beta pleated sheets and beta turns
  • The secondary structure of proteins is determined by the amino acid sequence
  • The roles of intra-chain hydrogen bonds are covered
  • Hydrophobic interactions and solubility are mentioned

Protein Tertiary Structure

  • Linking together secondary structural elements is explained
  • How smaller (β-α-β Loop) and larger (α-β Barrel) motifs and protein families are formed in proteins is covered

Protein Quaternary Structure

  • How particular proteins control metabolic pathways
  • The importance of primary, secondary, tertiary and quaternary structures is emphasized
  • The study of protein structures using molecular biology

Thermodynamics of Protein Folding

  • The top of the energy funnel shows that there are many possible protein conformations with high entropy
  • The bottom of the funnel shows that all of these conformations are reduced to a native shape

Sickle Cell Anemia

  • Hemoglobin single point mutation (E → V) causes misfolded proteins and misfolded erythrocytes
  • Description of normal and abnormal red blood cells, including the sickle-cell shape

Creutzfeldt-Jakob Disease

  • Prions are "proteinaceous infectious particles"
  • This disease involves the conversion of a normal protein (PrPc) into a disease-causing form (PrPSc)
  • The presentation highlights the key pathologic event in the disease

Conjugated Proteins

  • Properties and conventions associated with conjugated proteins are discussed (a class of proteins)
  • Examples of conjugated proteins (lipoproteins, glycoproteins, phosphoproteins, etc.) are mentioned
  • The prosthetic group characteristics of proteins

Protein Purification

  • Surface charge in proteins, methodologies for purification, examples are provided
  • Methods of controlling protein solubility, methodologies, examples and implications are discussed
  • Techniques that use hydrophobic nature, (hydrophobic interaction chromatography, reversed phase chromatography), are highlighted
  • Techniques that use molecular mass (size exclusion chromatography (SEC), ultrafiltration, SDS -PAGE), are included
  • Techniques of using bio specificity (post translational modification or engineering), affinity chromatography, are analysed
  • Combining different techniques for efficiency, methodologies are explored
  • Flow chart of protein purification
  • Protein purification balance sheet steps include:
    • Total protein content (TP)
    • Total activity (TA)
    • Specific Activity
    • Yield [% recovery]
    • Degree of purification

Protein Quantification

  • Colorimetric assays, spectrophotometric assays are explored
  • Methodologies for different quantitative analyses such as Bradford, Biuret, BCA, are covered
  • Absorbance based assays (methods using O.D. measures) are discussed
  • Typical applications for these colorimetric assays are presented, and common proteins used in assays are listed
  • A typical absorption graph showing standard curves

Chromatography

  • Different chromatography types including: ion exchange, hydrophobic interaction, gel filtration, and affinity chromatography, are discussed
  • Methods and applications for each type, including experimental setups and typical chromatograms

Electrophoresis

  • General principles of electrophoresis, including the use of stabilizing carrier matrices and separation based on charge, size, and shape
  • Detail of carrier matrices:
    • Agarose- based separations
    • Polyacrylamide-based separations
  • Staining and Quantification in electrophoresis applications
  • Discontinuous electrophoresis (disc electrophoresis)
  • SDS-PAGE and mechanisms of operation
  • Isoelectric focusing (IEF) and operational principles
  • Two-dimensional Electrophoresis (2D) methods
  • Capillary electrophoresis (CE) methods

Western Blot

  • This technique is discussed
  • The process and methods involving separation in a polyacrylamide gel is examined and visualised

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Explore intracellular protein purification steps, challenges in renaturation, and the importance of protease inhibitors and reducing agents. Discusses factors affecting protein stability and methods to minimize purification steps. Highlights aspects that impact protein loss or degradation.

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