Week 10 (Biochemical Methods I) Flashcards
What is the purpose of Blood tests?
- Measure Blood cholesterol levels
- detect Hormones
- Measure Blood sugar levels
What is the purpose of Urine samples?
-Detection of antibodies -Enzyme activity -liver function
How to separate a molecule of interest from all the other components: size
Separate large molecules from small molecules
How to separate a molecule of interest from all the other components: charge/polarity
- Separate molecules with opposite charges
- Separate highly charged molecules from less highly charged •Separate polar molecules from hydrophobic molecules
How to separate a molecule of interest from all the other components: Affinity/Specificity
Separate/identify molecules according to a specific interaction
How can molecules be separated by size?
- Gel filtration chromatography
- dialysis and other semi permeable membrane methods
- electrophoresis
What is Gel filtration chromatography also called?
Size exclusion chromatography Gel permeation chromatography
What do all forms of chromatography comprise?
- a mobile phase
- a stationary phase
Why does separation of molecules occur?
because they partition differently between the two phases (mobile and stationary)
Give an example of Chromatography theory
Molecule A has a high affinity for the stationary phase Molecule B has a high affinity for the mobile phase This allows effective separation of the two molecules
Which format do most chromatography techniques use?
Most chromatography techniques use a column format Mobile phase is a liquid fed into the top moves down the column Separation occurs as molecules move down the column
Chromatography terms: Column
Tube in which the separation process occurs
Chromatography terms: Matrix (resin/beads)
Material packed into the column that does 5e separating (stationary phase)
Chromatography terms: solute
mixture of molecules you’re trying to separate
Chromatography terms: solvent
Liquid in which solutes are dissolved (same as the mobile phase)
Chromatography terms: Eluant
What comes out of the column
Chromatography terms: fraction
Separate tubes the eluant is collected in
Chromatography terms: resolution
Degree of separation of different molecules
Gel filtration matrix
- Tiny little beads made of cross-linked polymers e.g. sephadex
- So tiny appear only as a fine white powder
- Mixed with water forms a slurry that resembles the texture of sand in water
Gel filtration matrix: What do the cross-linked polymers create?
A mesh-like porous structure within the beads
- Small molecules can fit inside these pores & enter the beads
- Large molecules can’t fit into these pores so are excluded from the beads
Gel filtration: stationary phase
beads and the spaces inside the beads
Gel infiltration: Mobile phase
Space outside the beads
How does gel infiltration separate molecules?
- Small molecules, which can enter the beads, enter the stationary phase
- Move slowly down the column
- Large molecules, can’t enter the beads, so can only be in the mobile phase
Gel infiltration: which molecules emerge first?
- Largest molecules emerge first
- Small molecules take longer
Gel filtration beads – different pore sizes
- A range of different gel filtration beads available
- Different size pores – exclude different size molecules
- Need to select the beads that have the best separating power for what you want to achieve
The applications of gel filtration: Preparative
- Purification
- De-salting
The applications of gel filtration: Analytical
- Determination of molecular mass
- Analysis of oligomer formation
Gel filtration in protein purification
Separation of a single protein from a mixture not easy - sizes too similar, overlapping elution of different proteins
-A final cleanup step to remove a contaminant of a different size, very commonly used
Describe De-salting by gel filtration
Removal of salt (or other small molecules) from a pure protein Large difference in size, so effective separation relatively easy & quick Protein is larger so comes out before the salt
Explain the determination of molecular mass by gel filtration
- Using a series of markers of known molecular mass -Plot elution volume against log molecular mass - should be a straight line
- Then run unknown test sample on the same column
- The point of elution of the unknown from the column can then be converted into a molecular mass
What is the critical assumption in the determination of molecular mass by gel filtration?
Critical assumption that all the proteins are the same
Explain the analysis of oligomerisation by gel filtration
- Gel filtration analyses a protein in its native folded state Dimers, trimers, tetramers remain bound together
- Will elute earlier than the monomer
- If you know the molecular mass of the monomer, can determine: if oligomers form what oligomers form (dimers, trimers….) the proportion of the sample that is oligomerised
What are the Advantages of gel filtration?
- Cheap
- Easy
What are the disadvantages of gel filtration?
- Slow
- Not very good resolution (unless large differences in size)
Improving resolution: The longer the column Is there disadvantages of having longer columns?
- the better the resolution
- Longer columns take longer to run
FPLC = Fast Protein Liquid Chromatography (FPLC) systems & automation
- Uses a pump to push the liquid through the column at desired rate
- Need to ensure pressure not too high, but significantly faster than by gravity flow
- Also automate the fraction collecting
- Various detectors to analyse the eluant as it comes out, tell you which fractions have protein in, which have salt in
What is HPLC?
High Pressure Liquid Chromatography or High Performance Liquid Chromatography
Explain HPLC
Use much finer/smaller beads
•less space around the beads – need much higher pressure to push liquid through the column
What are the advantages of HPLC?
- Fully automated
- Quicker
- Better separation
What are the disadvantages of HPLC?
Many proteins don’t like the high pressure as it can break the weak bonds in the protein
What is a Semi-permeable membrane?
- Small molecules can pass through the pores in the membrane -Large molecules cannot pass through the membrane
- This allows separation of the two molecules
Dialysis – what it looks like
- Dialysis tubing is formed from cellulose
- It is supplied as a flattened tube -If you wet it, the tube opens up
- Can form a sack to hold your sample by tying knots at the ends
- place the sack into a test tube with water to create a concentration gradient
- small molecules will diffuse out down the concentration gradient
- repeat until separated
What are the applications of dialysis?
Proteins: De-salting Buffer exchange
What are the disadvantages of dialysis?
- Slow
- Can cause proteins to precipitate out of solution
- Sample can get diluted
- Some sample may stick to the membrane
What are the advantages of dialysis?
- cheap
- easy
What is ultrafiltration?
Ultrafiltration occurs when fluid passes across a semipermeable membrane (a membrane that allows some substances to pass through but not others) due to a driving pressure
Suspended solids and solutes of high molecular weight are retained, while water and low molecular weight solutes pass through the membrane.
If you want to separate a large molecule, what would you change in the process of gel filtration?
You would use a resin with a larger molecular weight cut off
Improvements for dialysis experiments
Different dialysis tubing available with different size pores or molecular weight cutoffs MWCO)
Describe the process of dialysis
- Prepare a large volume of water/buffet
- Place the dialysis tubing containg the sample
- Allow equilibrium to be reached
- Not completely separated the two molecules
- Take dialysis tube ans put it into a fresh breaker to reestablish the concentration gradient
What is ultrafiltration?
- Semi permiable membrane at the bottom of the structure
- Sample loaded at the top
- Gas pressure provides driving force that
- Only small molecules cross the semi permeable membrane
Describe another variation of ultrafiltration
- Semi permeable membrane across the middle of the tube
- Sample loaded in the top
- Centrifugation used as the driving force that pushes the sample from the top top to the bottm
- Large molecules that cant cross the membrane left at the top
- Small molecules pass through the membrane
What are the risks associated with ultrafiltration?
-Possible to dry out the sample
