Lecture 3 Slides Purifying And Analyzing Proteins Flashcards
What does protein kinase phosphorylate
The hydroxyl group of a serine, threonine, or tyrosine
How are protein kinases used
Control activity and stability of target proteins
Regulate protein protein interactions
Signaling pathways
What does phosphorylation affect
Conformation of a protein and interactions with ligands
Histidine kinases
Signaling proteins found in prokaryotes, fungi, and plants
Protein Phosphatases
Perform reverse of protein kinase
Less specific than protein kinase with regard to substrate
Just as important for controlling activity of the protein targets
Can a protein be a target for multiple kinases
Yes. Each kinase would target a different amino residue
Cell fractionation
Centrifugation to separate cell components based on size, mass, density
Goals of using cell fractionation
Enrichment of organelles for protein purification
Cell free systems to study motility, enzymatic reactions, etc.
Fractionation methods 4
Break cell with osmotic shock, mechanical force, nonionic detergent
Plasma membrane and other membrane systems form vesicles
May need to include inhibitors of proteases, reducing agents
Some organelles remain intact
Cytoplasm
Contents of eukaryotic cell outside of nucleus
Cytosol
Contents of cytoplasm outside of membrane-bound organelles
How does cell fractionation by differential (rate-zonal) centrifugation work
Separation is based on size, density of organelles or large protein complexes
Cell homogenate is placed in tube
Slow centrifugation
Pellet contains whole cells, nuclei, cytoskeletons
Subject supernatant to medium speed centrifugation
Pellet contains mitochondria, lysosomes, peroxisomes
Supernatant subjected to high speed centrifugation
At cell free system, can use this supernatant for in Vitro translation
Pellet contains microsomes and small vesicles
Supernatant subjected to very high speed centrifugation
Pellet contains ribosomes, viruses, large macromolecules
Microsomes
Vesicles produced by fragmentation of endoplasmic reticulum when cell is organized
Velocity sedimentation
Particles are separated by size and shape; a density gradient prevents mixing. The gradient is formed prior to centrifugation.
S-value
Sedimentation coefficient - depends on Mass, volume, surface area of particle
Stages of velocity sedimentation
Place multi sediment sample in tube over stabilizing sucrose gradient
Centrifugation
A layer of fast sedimenting component forms under slow sedimenting layer. There is sucrose above, below, and in between layers
Fractionation
Example of velocity sedimentation. What do results mean?
Example of ribosomes and polyribosomes. You see 40s and 60s, and then 80s. You know this is a eukaryotes because of the 40 and 60. The polyribosome made by those two parts will be less dense, at 80, than th sum of their individual densities
Equilibrium density gradient sedimentation
Density gradient is formed mechanically (sucrose gradient) or by centrifugation (cesium chloride gradient); particles migrate to a point in the density gradient where they are in density equilibrium with th surrounding solvent.
Density gradient mechanical
Sucrose gradient
Density gradient by centrifugation
Cesium chloride gradient
In a sucrose gradient, in what order do fractions line up
Bottom to top, high buoyant density to low buoyant density
If you separate dna from RNA, which has higher density
RNA
How can you density separate different DNAs
By different base compositions (GC rich vs AT rich)
Column chromatography
Based on charge and size
An anion exchange column has positively charged matrix that interacts with negative charges on the proteins. Positively charged matrix will slow down negative particles as it interacts with them.
Two types of chromatography
Ion-exchange
Gel filtration
Gel filtration chromatography
Matrix of porous beads will trap and retard smaller molecules that can get caught in it, thus separating small from large molecules
What is ionic buffer
It is a charge that reacts with an opposite to slow it down
Affinity chromatography
Brads with covalently attached substrates bind specific molecule letting the rest pass
What can be bound to beads in affinity chromatography
Substrate for enzyme
Antibody
Ligand
-peptide, DNA fragments, metal ion (Ni, Cu)
How does eluting solution separate what is bound to beads in affinity chromatography
Eluting solution will contain component that decreases binding. For example, a binding competitor
SDS-PAGE
Sodium dodecyl sulfate pokyacrylamide gel electrophoresis
How does SDS PAGE work
Denature proteins by boiling them in SDS
Heat denatures proteins
Ionic detergent binds hydrophobic regions on proteins
Coats proteins with negative charge relative to size of protein
Beta-mercaptoethanol
Reducing agent breaks disulfide bonds within and between proteins
How does SDS gel electrophoresis work
Gel is in plastic casing.
Top has negative cathode,
Bottom has positive anode.
Ample loaded onto gel by pipette.
Choose, for example, two samples. One with two proteins joined by a disulfide bond and the other a single protein.
Heat with SDS and mercaptoethanol.
Protein complex splits into two proteins covered with electrons. Single protein does not break but is covered in negative charges.
The larger particles will bind more to gel and go down more slowly than smaller particles.
Two dimensional gel electrophoresis
First dimension separates proteins based on isoelectric point in a pH gradient net (proteins treated with reducing agent but no SDS!)
Second dimension, separates proteins by molecular weight - first dimension strip is subjected to SDS-PAGE
How do you separate proteins by isoelectric point
At isoelectric point, the protein has no net charge and therefore no longer migrates in the electric field. So, suppose a protein is positively charged, low pH. It will increase in pH until it reaches its isoelectric point. Sam for high pH protein but in opposite direction.
Why do isoelectric point to separate peotein by molecular weight.
Because they will be neutral and will both only differ by weight when in gel
What does phosphorylation change?
It results in a change in the isoelectric point of a protein.
How does phosphorylation happen
Protein kinases transfer a phosphate group from ATP to a target protein
What are targets for phosphorylation
Hydroxyl groups on:
Serine
Threonine
Tyrosine
How does mass spectrometry work
MS is used for protein identification
A single protein spot is excised from gel
Peptides are released by tryptic digestion and their masses measured using maldi-tof mass spectrometry
Graphs m/z(mass to charge ratio) to abundance (on y axis)
Protein sequence databases searched for matches with theoretical masses calculated for all trypsin related peptides
Gene is isolated and identified
How does MS-MS work
Mass spectrometer gives peptide masses
Each peptide is then further fragmented at peptide bonds
Masses of fragments measured on a coupled second mass spectrometer
The mass differences bet. Fragments can be used to construct a partial amino acid sequence. The data may allow gene ID, or provide the means for cloning the gene.
Post translational modifications can be detected
Proteomics
Identification of a collection of proteins expressed
- in a specific cell type
- in a specific organelle
- under a specific set of environmental conditions
- in response to disease
- identifies post translational modifications such as phosphorylation
- relative concentrations of proteins can be compared
Proteomics methods
- protein fragmented by trypsin digestion; peptides separated by liquid chromatography
- MS-MS of peptide fractions
- comparison of peptide sequences to predicted proteins from genome sequence
Two examples of clinical proteomics
- blood or cerebral spinal fluid, or urine samples are subjected to protein fractionation and MS-MS analysis
- “bio markers” - proteins associated with disease state are discovered by comparing samples from normal and disease samples
Where are antigen binding sites on antibodies
NH2 ends of light and heavy chains
Epitope
Antigenic determinant. Consists of 5 to 7 amino acids or 1-6 monosaccharides
How is strength of antibody binding defined
By affinity or association constant
(Inverse of dissociation constant)
Ka = [AgAb]/[Ag][Ab]
What is true at concentration of free antigen required to fill half the sites on the antibody
[AgAb]=[Ab]
Ka = 1/[Ag]
Units are liters per mole
Where are the hyper variable regions of light and heavy chains
Inside the binding site
Where are the variable regions of light and heavy chains
Outside of binding sites
How is antibody diversity generated
Somatic cell combination occurs during B lymphocyte development in the bone marrow
How does recombination happen for antibodies
VDJ recombinase selects specific V, D, J regions. Imperfect joining creates additional variability.
What kind of diversity exists in human antibodies
> 10^12 different antibodies
Is each antibody unique
Each B cell produces a unique antibody as a result of the recombination
Are there different types of immunoglobulins (antibodies)
Yes, depending on which C region is found on the heavy chain
Process to produce antibodies
- Purify protein of interest to homogeneity
- Inject antigen into animal
- Immune response - if the antigen binds to the body on a B cell, that cell is activated
- Test serum over several weeks for presence of specific antibody
- Recover antibodies from serum (polyclonal antibody)
How does immune response happen
- A naive cell, B lymphocyte is first exposed to antigen (in bone marrow)
- The cell creates memory cells and effector cells. More of the latter.
- The memory cells act during a second exposure to the antigen, and there is an even stronger response, with even more memory cells and effectors
Polyclonal antibody
Consists of many different antibodies, each produced by a different clone of B cells and each recognizing and binding to an epitope.
How many epitope a may a single antigen have
Many
How many epitopes will a polyclonal antibody recognize on an antigen
Many
Monoclonal antibody
Derived from a clone of cells from a single B cell producing a single type of antibody molecule
How is a monoclonal antibody produced
- A mouse is immunized with antigen X and a mutant cell line is derived from a tumor of B lymphocytes
- The mouse creates antibody.
- The mouse B lymphocytes die a few days after culture. The mutant cells grow indefinitely in a normal medium.
- Dead lymphocytes and living cells are fused.
- Fusion products played in multiple wells
- Only hybridomas grow on the selective medium
- Test supernatant for anti-X antibody and redistribute cells from positive well at ~1 cell per well.
- Allow cells to multiply then test supernatant for antibodies
Positive clons will provide a continuous source of antibodies
How many epitopes will a monoclonal antibody recognize
One
Multi silent antigen
Has multiple epitopes
Western blotting or immunoblotting process
Proteins from gel transferred to a membrane
Membrane is incubated with primary antibody against protein of interest
Unbound antibodies washed away
Membrane incubated with enzyme-linked secondary antibody against primary antibody
Unbound antibodies washed away
Product of enzyme reveals protein(s) bound by primary antibody
How are secondary antibodies marked
With colorimetric or chemiluminescent marker
Why use secondary antibody
It amplifies the signal and is conveniently available from a commercial source.
