Protein Purification(notinexam ) Flashcards
What can you do when studying proteins
What is a zwitterion and the isoelectric point
Why are proteins expressed recombinantly
How can you determine a specific amino acid from a ramachandran plot *
Look at the cluster of points corresponding to the amino acid residues of interest.
Compare these clusters to the typical regions for different secondary structures.
Note any residues in unusual positions, which might indicate glycine (broader distribution) or proline (restricted angles).
Specific Case Identification:
Glycine: Check for points that lie outside the usual allowed regions for most amino acids. If you see points in the top left quadrant (positive φ and ψ angles), it is likely to be glycine.
Proline: Check for points tightly clustered around φ = -65° and ψ = 150°.
Other amino acids: While it is more challenging to identify specific non-glycine, non-proline residues just from the plot, context from the protein structure or sequence can aid in identification.
How can you identify a salt bridge *
A salt bridge in proteins is an ionic interaction between oppositely charged amino acid side chains.
Give an overview of recombinant protein expression
What are the different choice of systems to express a protein in
What are the different choice of systems to express a protein in
What factors need to be considered when choosing an expression system
What are the different methods of extracting proteins from cells
How can different components of a cell be separated
What properties are separation techniques of proteins based on
How can you separate different components of a cell
How can proteins be separated using solubility
Give an example of size based separation of proteins
What methods can be used to separate proteins
How does gel filtration work *
Column Setup:
A column is packed with porous beads made of materials like agarose or polyacrylamide.
These beads have pores of specific sizes, allowing smaller molecules to enter the beads while larger molecules cannot.
Sample Application:
The sample mixture is applied to the top of the column.
The sample then flows through the column, typically under the force of gravity or using a pump.
Separation Process:
As the sample flows through the column, molecules diffuse in and out of the pores of the beads.
Larger molecules cannot enter the pores and are excluded, thus they move around the beads and travel through the column faster.
Smaller molecules enter the pores and have a longer path to travel, so they move through the column more slowly.
Elution:
The separated molecules elute from the column in order of decreasing size, with the largest molecules eluting first and the smallest molecules eluting last.
Elution is usually done with a buffer that maintains the integrity of the molecules.
How does affinity chromatography work *
Ligand-Matrix Preparation:
The chromatography column is packed with a matrix (e.g., agarose or sepharose) to which a specific ligand is covalently attached. The ligand is chosen because it has a strong and specific binding affinity for the target molecule. Examples of ligands include antibodies, enzymes, or small molecules.
Equilibration:
The column is equilibrated with a buffer that sets the appropriate pH and ionic strength to ensure that the ligand can effectively bind to the target molecule.
Sample Application:
The sample containing a mixture of molecules is applied to the column. Only the target molecule with high affinity for the ligand will bind to the ligand on the matrix, while other molecules will pass through the column without binding.
Washing:
The column is washed with the equilibration buffer to remove unbound and non-specifically bound molecules. This step ensures that only the target molecule remains bound to the ligand.
Elution:
The bound target molecule is eluted by disrupting the interaction between the target molecule and the ligand. This can be done using:
Competitive Elution: Adding a solution of a free ligand or an analogue that competes with the immobilized ligand for binding to the target molecule.
Change in pH or Ionic Strength: Altering the buffer conditions to weaken the interaction.
Chaotropic Agents: Using substances that disrupt the binding by affecting the structure of the target molecule or the ligand.
The elution buffer is carefully chosen to maintain the integrity and activity of the target molecule.
How does affinity chromatography work *
What is SDS-PAGE
What is SDS-PAGE
How does SDS-PAGE work
How are the proteins detected in electrophoresis
How does western blotting work*
After electrophoresis, the separated proteins are transferred from the gel onto a membrane (usually nitrocellulose or polyvinylidene fluoride (PVDF)). This is done using an electric field in a process called electroblotting. The proteins move out of the gel and bind to the membrane, preserving the pattern of separation.
Blocking:
The membrane is incubated with a blocking buffer (commonly containing non-fat dry milk or BSA) to prevent non-specific binding of antibodies to the membrane. This step reduces background noise.
Primary Antibody Incubation:
The membrane is incubated with a primary antibody specific to the target protein. This antibody binds to the protein of interest on the membrane.
Washing:
Excess primary antibody is washed off with a washing buffer (usually containing a mild detergent like Tween-20).
Secondary Antibody Incubation:
The membrane is then incubated with a secondary antibody that binds to the primary antibody. The secondary antibody is conjugated to an enzyme (e.g., horseradish peroxidase (HRP)) or a fluorescent dye to facilitate detection.
Detection:
The presence of the secondary antibody is detected using a substrate that reacts with the enzyme to produce a detectable signal. For HRP, common substrates include chemiluminescent substrates (e.g., ECL) that emit light when oxidized. The signal can be detected using various methods:
Chemiluminescence: The emitted light is captured on X-ray film or by a digital imaging system.
Fluorescence: Fluorescently labeled antibodies are detected using a fluorescence scanner.
Colorimetric Detection: Enzymatic reaction produces a colored precipitate on the membrane, which can be seen directly.
What are the different western blot detection methods
What are the different western blot detection methods
How does 2D electrophoresis work
What is isoelectric focusing
What is the second step of 2D gel electrophoresis
How can you analyse proteins using fragmentation
How can proteins be fragmented enzymatically
How can proteins be fragmented chemically
How can proteins be fragmented chemically
How can proteins be identified using mass spec
What is the workflow of protein sequencing using mass spec *
Protein Extraction and Purification: Isolate proteins from biological samples.
Protein Digestion: Enzymatically digest proteins into peptides.
Peptide Separation: Separate peptides using liquid chromatography.
Mass Spectrometry Analysis: Ionize peptides and analyze their m/z ratios; perform MS/MS for fragmentation.
Data Acquisition: Collect mass spectra of precursor and product ions.
Data Analysis: Identify peptides and proteins using database searching or de novo sequencing.
Validation and Quantification: Validate identifications and quantify protein levels.
Bioinformatics Analysis: Interpret biological significance and integrate data.
What is the workflow of protein sequencing using mass spec *
How can protein crystallography be used to solve protein structures
How does protein crystallography work*
Protein Purification: Obtain highly pure protein.
Crystallization: Find and optimize conditions for crystal growth.
Crystal Harvesting: Carefully mount crystals for data collection.
Data Collection: Collect X-ray diffraction data from crystals.
Data Processing: Process diffraction data to obtain structure factor amplitudes.
Phase Determination: Determine phases using MR, MIR, or MAD/SAD methods.
Electron Density Map Calculation: Calculate the electron density map.
Model Building and Refinement: Build and refine the atomic model.
Validation: Validate the accuracy of the model.
Structure Analysis and Interpretation: Analyze and interpret the protein structure.
Deposition: Deposit the structure in the PDB.
How are protein crystals grown*
Vapor Diffusion:
Hanging Drop Method: A small droplet containing the protein solution is mixed with a precipitant solution on a coverslip. The coverslip is then inverted over a well containing a reservoir solution with higher precipitant concentration. Over time, water vapor diffuses from the droplet to the reservoir, gradually concentrating the protein and precipitant in the droplet, leading to crystal formation.
