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
