Chapter 5: Methods in Protein Biochemistry Flashcards
What is the Proteome?
The entire collection of proteins found in an organism
How is Cell Fracturation prepared?
- Cells are broken which produces cell extracts (homogenates)
- Cell suspension prepared by mincing via 1/3 ways
a. Sonication - disrupts cell membranes through vibrational ultrasonic waves
b. Shearing - forcing cells through small opening
c. Incubation with mild detergents - disrupts cell membrane
Once the Cell Fracture has been prepared how is centrifugation carried out?
- Once target protein is isolated it undergoes preparative centrefugation
- Four fractions containing Nuclei, mitochondria, plasma membrane, and cytosol
- An estimate for the total amount of protein can be made by measuring sample absorbance at 280 nm
Why is absorbance measured at 280 nm?
Because it is the wavelength where tryptophan most absorb light
What is salting out?
A process to separate proteins
- Add increasing amounts of saturated salt solution to form ionic interactions with water
- Results in insoluble aggregates of proteins
- Often use (NH4)2SO4
What is dialysis?
- A process of removing a salt (ammonium sulfate) from the protein sample
- small pores in tubing which allow salt to diffuse out but protein stays trapped
What is Column Chromatography?
A process which separates proteins based on chemical or physical interactions
- A column with a matrix in combination witha continuous flow of buffer causes separation of protein
- Measure protein by 280 nm
- Fractions are then collected in small containers
What is Gel Filtration Chromatography?
- Also called size exclusive chrom.
- separates proteins by size
- Beads have pores which means there is selectivity for which proteins travel
- large proteins travel through while small proteins get stuck(delayed) in carbohydrate beads
How does protein size factor into elution profile?
The larger the protein the faster it elutes
What is HPLC?
- High Performance Liquid Chromatography
- Column contains very small beads leading to better separation
- High pressure needed to force buffer and protein through column
- Expensive
What is Ion-Exchange chromatography?
- Uses difference in charges of proteins
- Can have anion or cation charged matrix
- Charged beads will allow same charged proteins to travel freely while oppositely charged proteins are bound until elution buffer is run.
What is Affinity Chromatography?
- ## Uses the binding properties of a target protein to separate it from proteins that lack the same properties
What is Ni-Affinity Chromatography?
- Protein starts or ends with 6 His
- Binds to Ni
- Elute with Imidazole buffer
Rank the efficiency of purification of Chemical methods
- Preparation of a crude cell extract
- Subcellular fraction using centrifugation
- Ammonium sulfate precipitate
- Gel-filtrate Chromatography
- Ion-Exchange Chromatography
- Affinity Chromatography
What is Gel Electrophoresis?
- Cheap
- Apparatus is used to determine protein purity
- provides an approximate mass of proteins (subunit composition)
- PAGE (Polyacrylamide gel electrophoresis)
- PAGE = separates proteins based on charge
Describe the setup of a PAGE electrophoresis and what it does.
- Electric field pulls proteins according to charge
- Gel matrix hinders proteins based on size and shape
- Velocity = (E*z)/f (f = 6pi)
What is SDS-Page
- SDS is a detergent that adds net negative charge to each protein to aid in the migration toward the anode
- Provides uniform charge
- Denatures shape so only size is accounted for
- Small molecules move the fastest
What is Isoelectric Focusing?
- No SDS needed
- Gel matrix has pH gradient
- Protein migrates until they are uncharged when pH = pI
What is 2D Gel Electrophoresis?
Separation of proteins by pI then SDS page (sorts by molecular mass)
How does Edman Degradation work?
- PITC treated with trifluoroacetic acid
- This cleaved b/t first and second peptide
- Thiazolinone derivative is made and the amino acid chain loses an amino acid
- Proton is added to N
- Process repeats ( Treat next aa with PITC and base and repeat)
- Only works for up to 50 aa
What are the differences between Matrix-assisted laser ionization and Electrospray ionization?
Matrix-assisted laser desorption ionization (MALDI)
- Laser pulse ionizes sample in absorbing matrix
- Collision with protein and transfer change occurs
- Detector in mass spectrometer measures acceleration of ions determining their mass
Electrospray Ionization (ESI)
- Polypeptide fragments (usually tryiptic fragments which is fragments of a trypsin cleaved protein) are released via high voltage metalic capillary
- Conditions cause solvent containing peptides to evaporate
- This makes highly charged gas molecules
What is X-ray Crystallography and what are the steps?
- A method of protein structure determination based on diffraction of X-rays by protein crystals
Steps:
1. Purify protein
2. Grow diffraction quality crystals
3. Determining the phases of diffracted X-rays
4. Calculate electron density map
5. Fit amino acid sequence into density map
What is Cryo-electron Microscopy?
- A form of microscopy that is good for larger protein complexes
- Compiles 2D projections to generate high resolution 2D images then can be built into 3D model
How many and what type of chains can B cells create?
- B cells can make two classes of Ig light chains (λ and κ) and five classes of Ig heavy chains (µ, α,γ, δ, and ε)
What is an Epitope?
- A specific site on antigen antibodies can bind to
- Example: protein from Borrelia burgodorferi causes Lyme disease and has several epitopes that antibodies can recognize
Define and compare monclonal and polyclonal antibodies
Monoclonal:
- Homogeneous immunoglobin species that recognizes ONE epitope on an antigenic protein
- Isolated from hybridomas
Polyclonal:
- Heterogeneous mixture of immunoglobulin proteins that recognize ONE OR MORE epitopes on an antigenic protein
- Isolated from blood
How are polyclonal antibodies generated?
- Immunization occurs
- Blood sample is obtained containing nonspecific and specific antibodies
- Purification via affinity chromatography
- Three fractions are collected (flow through, Nonspecific antibodies, and affinity purified polyclonal antibodies
How are monoclonal antibodies generated?
- Multiple immunizations
- B cells are isolated
- B cells are joined with immortalized tumor cell
- Antigen specific and nonspecific hybridoma are generated
- Antigen specific hybridoma identified via screening
- Cloning of hybridomas
- Tissue is cultured containing antigen specific monoclonal antibodies
What is Western Blotting?
- Detects proteins with high sensitivity
- Used to detect proteins separated by SDS-PAGE
- Uses two antibodies
- Primary binds to target protein
- Secondary contains enzyme for visualization
What is ELISA?
- Enzyme linked immunoabsorbant Assay (ELISA)
- Detects proteins with high sensitivity
- Identifies low level antigenic proteins in solution
- Used in samples such as plasma
What is immunoprecipitation
- Variation of affinity purification
- Monoclonal antibody covalently linked to carb. bead
- Incubation of protein mixture and MAb-beads which are collected by centrifugation
