Section 2 Flashcards
What are the physical and chemical properties of proteins?
used for purification and analysis
Mass or size (and shape)
Density
Electrical charge
Binding affinity
What are the 3 main separation methods?
- Centrifugation
- Electrophoresis
- Chromatography
How does centrifugation work?
Particles move depending on their density relative to the density of the fluid
Denser particle than the fluid go to the bottom and for a PELLET, less dense come up to the surface, same density don’t move (particles start dispersed)
What unit is used to measure centrifugal force?
earth gravity in g (abt 10 g)
What are 2 types of of centrfugation set ups?
Swinging bucket rotor: bucket start vertical and swings to horizontal when spins
Fixed-angle rotor: bucket fixed at 45˚ angle
What determines the rate at which the supernatant is cleared of particles at given centrifugal force?
For particle of similar shape, it is determined by the size/mass
“Size” unit calculated this way called Svedberg or S
What is Differential centrifugation?
It is the fact of using different centrifugation speeds to recover different particles
What fluid is usually used in a centrifugation tube?
Sucrose density gradient
Particles of interest stop at a certain specific gradient (layer) in the tube corresponding to their density
What characteristic of proteins is electrophoresis based on?
Charge : mass ratio
Speed of migration determined by net charge/mass ratio
Direction of migration depends on net charge (- go towards + end and inversely)
What is SDS used for?
SDS = anionic detergent sodium docecyl sulfate
Its a hydrophobic tail (12 C) with suflate at one end, one of the O- coupled with Na+
SDS denatures proteins by interaction of hydrophobic tail with hydrophobic amino acid side chains (also binds to itself so coats the polypeptide chain)
Bc all negative, various parts of SDS polypeptide chain repel each other which unfolds the protein
SDS also separated chains of multimeric protein → individual denatured polypeptides
Used in gel electrophoresis
When SDS denatures proteins, there is no difference in shape which could affect the mvt in gel
What is the isoelectric point of a protein?
Its the pH at which the sum of all charges = 0
Low pH = more protons available = more + charges
High pH = less protons = more - charges
Acidic residues are neutral in low pH and negative in high pH (ex: COOH → COO-)
Basic residues are neutral in high pH and positive in low pH (ex: NH3+ → NH2)
The isoelectric point depends on the amino acid composition of each protein
What is isoelectric focusing?
It is a type of electrophoresis
A pH gradient is established using special buffers (ampholytes) immoblized in acrylamide gel
ex: cathode (+) pH 2 → pH 10 anode (-)
Proteins migrate towards their isoelectric point
acidic proteins migrate towards acidic pH, towards the cathode and inversely
Proteins resolve in narrow stripes at each isoelectric point
Explain 2-dimensional gel electrophoresis
- Isoelectric focusing:
All proteins resolve into stripes (separation by charge)
Turn stripes horizontally at the top of an SDS electrophoresis - SDS PAGE (separation by size)
Final result = dots on 2D sheet, helpful bc no relation between pI and molecular weight so reveals simultaneously multiple different proteins
What is the downside of Mass spectrometry?
you can’t re-use the particles as they are destroyed in the process
Which 3 concepts are the basis of mass spectrometry?
- Produce dispersed ions in a gas phase
- Measure acceleration of the ions in electric or magnetic field
- Acceleration depends on mass/charge ration (m/z)
*Molecular weight is specific to each molecule, if charge = 1, m/z = MW
What is a commonly-used process for generating gas-phase ionized molecules ?
Electrospray ionization:
Tranfer of ions from solution into gas phase before they are subjected to mass spectrometry
Produces gas-phase ions
What is the role of the mass analyzer?
Separates the ions according to mass/charge ratio
What is MS/MS or tandem MS?
It is the principle of recovering an ion, fragmenting it by high energy collision with inert gas
doing mass spectroscopy on the fragments
(double mass spec)
2nd dimension (MS) gives info on amino acid composition
Where does fragmentation happen in MS? (on the polypeptide)
At peptide bonds
MS-MS doesn’t break every peptide bond → partial and random process
Partial = on average only one (or a small number) of peptide bond/molecule
What is proteomics?
Proteomics is the analysis of biological protein samples by mass spec and bioinformatics (computer analysis of DNA and protein sequences) in order to identify the population of proteins present in given subcellular organelle
Identification of all polypeptides in complex sample
How does chromatography work?
Separation of components based on differential interaction with a immobile solide material
+ interaction with solid phase = slower moving down
- interactions with mobile phase = faster moving down
What is gel filtration chromatography?
Separation based on size
Solid phase get bead have pores so that only small proteins can pass through (way slower)
Large proteins go through faster
Beads = polymer get beads
How does ion-exchange chromatography work?
Separation based on electric charge
Based on positively charged character of beads
- Negatively charged proteins stil to gel beads, +vely charged proteins are repelled and go down fast
- Elute negatively charged protein with NaCl solution (Cl- replaces anions bc stronger, Na+ replace +ive proteins if the beads were negative) –> «ion exchange»
What is an antibody?
A protein that recognizes, by highly-specific binding, a target molecule
It recognizes the epitope on the anitgen
How does antibody-affinity chromatography work?
Affinity dependent on pH
Can isolate 1 single particular protein in a complex mixture with antibody recognition
1. Antibodies specific for the targetted protein can be COVALENTLY coupled to the solid phase
2. Insert protein mixture and pH buffer mobile phase
3. Wash with pH 7 buffer, every protein comes down except the targeted one that interacts non-covalently with the antibody
4. Elute with pH 3 buffer to inactivate antibody and collect the targeted protein
What are different types of chromatography?
- Antibody-affinity chromatography
- Ion-exchange chromatography
- Gel filtration chromatography
What regions do primary and secondary antibodies recognize?
How do we call immunodetection using both?
CDR (complementarity-determining region) at tip of the light chain of primary antibodies recognize epitope on antigen
Secondary antibodies recognize the constant region of the primary antibody
2nd Ab is a «universal» reagant, buy for cheap an anti-anti-mouse (bc all anti-mouse primary antibodies have same cste region)
We call it indirect or «sandwich» immunodetection
How does Immunoblot (Western blot) work?
- Separate complex protein mixture by SDS polyacrylamide gel electrophoresis onto a membrane
- Use primary antibodies to recognize individual protein species
- Use secondary antibodies for detection (and to amplify the signal)
Can use the membrane and treat it with different antibodies to detect different proteins in different stripes
What is Immunoprecipitation useful for?
How does it work?
Used to isolate a protein COMPLEX (with its antibodies, partner proteins, etc.) from a protein mixture by using an antibody specific for that complex
ex: red and green exist as heterodimers, add antibody specific for red protein, introduce bacterial protein/2nd antibody th
What is the difference between immunoprecipitation and co-immunoprecipitation?
IP = protein carrying the epitope recognized by antibody
Co-IP = protein carrying the epitope recognized by antibody + any partner proteins stably associated with that epitope carrying protein (forming a complex to with the antibody binds)
What is Immunofluorescence microscopy?
Made by chemical coupling of a fluorochrome to the secondary antibody to allow detection in fluorescence microscope
- Prepare sample on microscope slide
- Incubate with 1ary Ab + wash away unbound Ab
- Incubate with fluochrome-conjugated 2ary Ab + wash away unbound Ab
- Mount specimen and observe
*can be used to see dirpersion of proteins in a cell (ex: GLUT2 present on lateral and basal plasma membrane surface, but not at apical surface)
What are 2 uses for genes encoding GFP (Green Fluorescent Protein) introduced into a cell ?
When introduced into cells of essentially any organism, GFP is produced in the living cells
- can be used as a reporter gene for transcriptional control elements
- can be made into fusion proteins to study intracellular protein localization
What is the structure of the gene coding for GFP fusion proteins to study intracellular protein localization?
Control Region (promoter) - natural protein-coding sequence - GFP encoding sequence
fusion protein = real protein + GFP at the end
What is the structure of the gene coding for GFP as a reporter gene to reveal gene promoter-driven transcription patterns?
Regulatory sequence (gene’s promoter) - Reporter gene (encoding for GFP or luciferase)
What is Kd?
the dissociation constant
