What are the cell dimensions of eukaryotic cells?
10-100 um
What are the cell dimensions of intracellular organelles?
0.01-10um
What are the main biomolecules?
proteins, rna, and dna
What is the function and dimensions of proteins?
They are the molecular machines involved in all cell functions.
2-20nm
What is the function and dimensions of dna?
They store information on the structure of all proteins.
Diameter - 2nm
What is the function of rna?
They transfer DNA to protein, protein synthesis, regulate of gene expression, and catalysis.
What is the flow of genetic information (central dogma)?
DNA to mRNA to protein
What is the largest intracellular organelle?
nucleus
As particles move to the bottom of a tube during centrifugation, the centrifugal force acting on them...
increases
What forces are important for particle sedimentation during centrifugation?
friction and buoyancy
What happens to particles in solution during centrifugation?
They move towards and away from the rotation center.
The friction force acting on a solid particle slowly moving in a liquid depends on...
particle shape, size, and the interaction between molecules of the liquid
Sedimentation velocity of thin linear particles is...
not dependent on the particle length
Sedimentation velocity of spherical particles is...
proportional to square of particle diameter
As particles settle, will particle density be more or less than solution density?
Particle density will be greater.
What is sedimentation velocities?
when particles are separated based on different velocities
Sedimentation velocity can b increased by increasing what?
g (gravity)
Friction coefficient f depends on what?
size and shape
Denser particles of the same size and shape move...
faster
What affects both volume and friction coefficent?
shape and size
True or false. Sedimentation velocity is proportional to the ratio of particle volume and friction coefficient.
true
Bigger spherical particles of equal density move what?
faster
For sedimentation of biological particles, is the dependence on size more important than the dependence on density?
Yes
Velocity-dependant seperation of near spherical biological particles is determined by what?
size differences
Bigger biological particles have what?
larger sedimentation velocities
The thin linear particle shape is common for what?
short double-stranded DNA, denatured RNA, denatured proteins, and cytoskeleton filaments.
Can thin linear particles be separated by length?
no
Linear thin particles of equal radius but different length have the same what?
sedimentation velocity
what is the Svedberg coefficient?
the sedimentation velocity normalized by the centrifugal acceleration
What does "s" depend on in the svedberg coefficient?
Particle properties. (ribosomes are measured in s unit)
What is the organelle extraction by differential centrifugation?
there are several cycles run with increasing rotation speed and duration, after each run the largest particles will form a pellet at the bottom of the tube and it will be removed.
Would particles with different sedimentation velocities
form
separate bands?
No
What is the density gradient?
its when heavier fluid goes on top and will eventually sink due to gravity
What is convection?
the flow and mixing of the suspension caused by density gradients in the centrifugation field.
What is rate zonal centrifugation?
it is centrifugation in a density gradient. When a particle mixture is placed on top and a solution of sucrose is selected so that particle density is greater than solution density.
What happens in centrifugation in a density gradient?
When particles move at a different speed, they form discrete bands, which are determined by sedimentation speed, which is dependent on particle size, density, and shape.
Isopycnic centrifugation
When a particle floats or sinks to its equilibrium point where particle density equals the density of the solution.
What does isopycnic mean?
same density
Svedberg coefficient depends on what?
particle size, shape, and density
The purpose of a density gradient in the Rate Zonal centrifugation is to what?
prevent convection
The most important factor responsible for cell separation in Rate Zonal centrifugation is cell?
size
Isopycnic centrifugation separates particles according to their differences in...
density
Which forces determine electrophoresis of bio-molecules?
electrical and friction
Electrophoretic velocity of short DNA molecules in water-based electrolyte is?
not dependant on molecule length
Gel is solidified for DNA slab electrophoresis in order to...
prevent convection
During electrophoresis, DNA molecules move towards what?
the anode
During electrophoresis, native protein molecules move toward...
anode or cathode
SDS-PAGE separates proteins based on differences in protein...
amino acid chain length
Which molecules can be separated by electrophoresis in water-based electrolyte (without gel)?
non denatured RNA, non denatured proteins, and denatured proteins
What is electrophoresis?
movement of electrically charged particles in a liquid medium caused by electric force
If particles move with different velocities they do what?
they become spatially separated
The separation factor ratio depends on what 3 properties?
1. charge
2. shape
3. size
What is the source of DNA charge?
phosphate groups
If DNA is negatively charged then the charge is what?
distributed uniformly
How does the short double stranded DNA straighten out?
when the charged groups repulse one another
what is the shape of short dsDNA?
linear or rod shaped
Is separation of short dsDNAs of variable length in water-based electrolyte possible?
No
EP of short dsDNA in a gel.
Double-stranded DNAs within a certain length range can be separated by EP in a gel based on their length differences
What is the the gel?
A mesh of fibers forms pores filled with electrolytes through which DNA molecules can move. The gel can be liquid or solid.
True or false. Longer molecules move through gel slower than shorter molecules
True
What is slab gel EP?
when a slab of gel is solidified to prevent convection. This occurs when samples are loaded on the cathode side and negatively charged DNA move towards the anode.
What is the field strength of gel?
5-20 V/cm
Length measurements are done by what?
DNA ladders
What is capillary gel EP?
It is run in thin capillaries so that gel can be liquid and require no convection and it is rapid dissipation of resistive heat.
Under denaturing conditions, EP separation of ssDNAs or RNAs
is
determined by what?
their length
What is the length speration range of EP gels?
function of gel pore size which depends on gel type and concentration.
polyacrylamide gel
small pores, length less than 1000 bp, and high senstivity
agarose gel
large pores, length less than 50,000 bps, and low sensitivity
the net charge of a native protein can be what?
positive, negative, or either
What is the most common method for electrophoretic protein separation?
SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)
Steps of SDS-PAGE
1. denature proteins
2. treat with SDS which coats proteins with negative charges
3. repulses negative charges by straightening out proteins
4. treat proteins behave like short dsDNA
SDS-treated proteins move toward the what?
anode
True or false. Protein charge is proportional to the protein length
true
SDS PAGE data presentation
Using a protein ladder and molecular weight
The level of (de)protonation depends on what?
the solution pH
When pH decreases and H increases what happens to protein charge?
it increases
When pH increases and H decreases what happens to protein charge?
it decreases
true or false. Each protein has its own unique pl value
true
What is isoelectric focusing?
Separation of native proteins based on differences in their pl values
A stable gradient of pH is created in what?
a gel
Where is the anode located?
low pH - positive
Where is the cathode located?
high pH - negative
True or false. IEF is independent of protein MW
true
When a molecule reaches its pl point what happens?
its charge becomes 0 and it stops
2D EP happens in what 2 steps?
1. IEF - isoelectric focusing
2. SDS-PAGE
Charge of which molecules is affected by changes of solution pH typical for intracellular environment?
Native proteins
In the Isoelectric Focusing method, the cathode ( (-) electrode) is placed in the region with?
high pH
The "housekeeping" RNAs are used in Northern blotting for...
measuring relative quantities of target RNAs
The role of albumin in Western blotting is to...
block non-specific antibody binding
The nitrocellulose membrane used in Western blotting binds...
all proteins
Which protein property makes it a candidate for the role of a “housekeeping” protein in Western blotting?
stability of protein quantity
How do we identify specific bio-molecule?
through staining with light/raditaion labels
What are the DNA/RNA labels?
hybridiziation probes
What are the protein labels?
immunistaining probes
What are the 3 blotting methods?
1. Southern - DNA
2. Northern - RNAs
3. Western - proteins
What are the blotting steps?
1. Gel electrophoresis
2. Transfer of molecules onto a solid
membrane
3. Staining, visualization and analysis
what is the hybridization probe
a bio-conjugate of cDNA and reporter molecule
Hybridization steps
1. target DNA/RNA and probe are denatured
2. The probe is added and binds to the target
3. light/radiation is detected
Northern blotting
detects a specific mRNA in a sample gene expression
What are the steps of northern blotting?
1. denature
2. separate by gel EP
3. Transfer to a membrane
4. Hybridization with probe
5. visualize
Relative mRNA amounts from different samples are measured by comparing what?
band intensities
true or false. mRNA is unstable
true
Immunolabeling probe - Western blotting
a bio-conjugate of antibody and reporter molecule
Western blotting steps
1. SDS PAGE
2. transfer proteins from gel to membrane
3. block antibody binding to membrane
4. label proteins with a primary antibody
5. label proteins with secondary antibody
6. detect the protein by optical imaging
examples of housekeeping proteins
actin, tubulin, and GAPDH
Genomics
analysis of organisms’ entire genomes or a large number of genes
Functional genomics
global analysis of gene expression; identification and quantitative measurements of all (or many) mRNAs
Proteomics
global analysis of cell protein content; identification and quantitative analysis of all (many) proteins
What is chromatography
A family of techniques for rapid separation of complex mixtures
of
molecules - fastest separation method
2 phases of chromatography
1. stationary phase (gel, liquid, paper)
2. mobile phase (liquid or gas)
Ion exchange chromatography
Stationary - resin beads with covalently bound charged molecules
mobile - an electrolyte buffer with a certain ionic strength and pH
WHat are the resin types in ion exchange chromatography?
anion exchanger- the resin has positive charges, it binds anions
cation exchanger-the resin has negative charges, it binds cations
What is elution?
step when main molecule separation takes place
Ionic elution
Increase salt concentration and protein molecules are displaced by negative or positive ions.
Separation is determined primarily by protein charge
pH elution
Increases or decrease pH toward protein pl
Separation is determined primarily by protein pl
Molecular exclusion chromatography
Stationary phase- porous gel beads
separation is based on molecule size
In cation exchange chromatography, what happens to protein charge?
it decreases
In anion exchange chromatography, what happens to protein charge?
it increases
Partition chromatogrpahy
stationary phase - a thin liquid film formed on the surface of an inert solid support
mobile phase - another liquid solvent
Paper chromatography
stationary phase - bound h2O
mobile phase- another solvent less polar than h2O (solvent moves up sheet using capillary forces)
Affinity chromatogrpahy
stationary phase - contains ligands immobilized on beads that bind a targeted protein
Separation is based on specific protein-ligand affinity
Absorption chromatography
stationary phase - a solid adsorbent (silica gel (SiO2) or alumina (Al2O3)
mobile phase - typically, liquid (e.g., ether, ethanol), or gas