Unit 3 - Protein analysis Flashcards
What are a diverse and rapidly growing segment of the prescription drug market?
Protein drugs
Which molecules are central to life and very important in biomedical research?
Endogenous proteins
- ones already in the body
How are target proteins harvested following microbial cell lysis?
(Host microbe grows target protein which is then harvested)
Chemically created channels
allowing proteins to leak
out through the cell membranes.
- Toluene, ether, dimethyl sulphoxide (DMSO), methanol, also used, detergents, enzymes, alkali, chelates and chaotropic agents
- Effective, but introduce additional chemicals into the lysate that can require later (downstream) removal from the purified materials.
Enzymatic permeabilisation
- Enzymatic permeabilization with agents such as lysozyme is used for E. coli, which has tough cell walls. Generally slower, and these added proteins could cause separation problems downstream.
Mechanical methods
- Mechanical methods The cell envelope is broken physically, and the intracellular components are released into the surrounding medium.
- Ultrasonication, freeze–thaw and liquid shear
Preferred as less clean-up is needed downstream, as no addition of reagents
Which chemicals are used to create channels to allow proteins to leak out of a microbe through the cell membranes?
Toluene Ether Dimethyl sulphoxide (DMSO) Methanol Detergents Enzymes Alkali Chelates Chaotropic agents
What are the advantages of chemically created channels?
they’re Effective
What are the disadvantages of chemically created channels?
Introduces additional chemicals into the lysate that can require later (downstream) removal from the purified materials
What is used to break down cell walls of microbes to release proteins?
Lysozyme is used for E.coli which has tough cell walls
What are the disadvantages of using enzymatic permeabilisation to release proteins?
Slower
Added proteins could cause separation problems downstream
How do mechanical methods release proteins from microbes?
Cell envelope is broken physically Intracellular components are released into the surrounding medium - ultrasonification - freeze-thaw - liquid shear
What are the advantages of mechanical methods to release proteins from microbes?
Less clean up needed downstream
- no addition of reagents
What is lysate?
Material obtained after cell lysis
Why does crude lysate need separation, identification and quantitation?
Very impure
- especially if extraneous chemicals used to cause lysis
How are proteins different to small molecules in terms of analytical methods?
Size
- generally macromoles
Chemical nature/functionality
Proteins adopt secondary/tertiary configuration
Lability (quality of being likely to change)
- many proteins are susceptible subtle changes or degradation
- can affect or negate pharmacological action
- whichever lysis, separation and analytical method is used
- target protein must remain unaltered
Give some examples of protein analysis methods
Fast Protein Liquid Chromatography - FPLC Mass Spectrometry - MS Electrophoresis Western Blotting - IHC - ICC
What is FPLC?
Fast Protein Liquid Chromotography
FPLC is a form of liquid
chromatography used to separate
or purify proteins from complex mixtures
What is Fast protein liquid chromatography (FPLC) Used for?
To separate or purify proteins from complex mixtures
What is the difference between HPLC and FPLC?
Particle sizes are larger in FPLC
- beads
Selective stationary phases
Low pressure
Glass or plastic columns
- The main interest is in the nature of the stationary phases used to achieve purification
What are the three main modes of FPLC?
Ion exchange - some selectivity based on ionic affinity Gel filtration - selective based on molecular size Immunoaffinity - highly selective - based on immuno-recognition
What is ion-exchange chromatography? (a form of FPLC)
Separation of ions (proteins) based on charge affinity
Molecules have affinity for counter exchange on stationary phase
How does ion exchange chromatography work?
Molecules have affinity for countercharge on stationary phase (ion exchanger)
- cationic exchange resin is negative (e.g COO-, SO3-) and
exchanges positively charged ions (cations)
anionic exchange resin is positive
and exchanges negatively charged ions (anions)
How does anion(positive) exchange chromatography work?
Protein mixture added to top of column
-ve charged protein retained as its attracted to stationary phase.
+ve charged/neutral protein eluted as its not attracted
Mobile phase changed - stronger eluting power - buffer pH -ve charged protein eluted because there's no affinity therefore proteins have been separated
How does gel filtration chromatography work?( a form of FPLC)
Molecules are separated based on size
Glass column filled gel which acts as the stationary phase.
The gel is made up of porous beads
- channels of predetermined size
Molecules larger than pore size cannot enter the pores and elute together as the first peak in the chromatogram
- total exclusion
Molecules that can enter the pores will have an average residence time in the particles that depends on the molecule’s size and shape
Different molecules therefore have different total transit times through the column
- selective permeation region
Molecules smaller than the pore size can enter all the pores - longest residence time on the column as they stay longer within the pores and elute as the last peak in the chromatogram
How is the stationary phase selected in gel chromatography?
Based on a knowledge of analyte mass
- available gels with defined upper and lower pore size
Which gels give a higher resolution in gel filtration?
Smaller range of pore sizes
- smaller range over which it can separate macromolecules on the basis of their size
Which gels give a lower resolution in gel filtration?
Wider range of pore sizes
- will permit fractionation of a larger range of sizes as an initial step when the approximate molecular mass is unknown
