Experimental techniques Flashcards
How are proteins extracted from a cell?
extract the protein in a buffer solution or of ionic strength compatible with that found inside the cell.
Add in:
enzyme inhibitors
EDTA/EGTA - reduce oxidation and inhibit metalloproteinases
DTT/2-mercaptoethanol - reduce oxidative damage
Glycerol - stabilisation
How are cells disrupted?
By physical means such as a homogeniser, blender or press them.
Also use enzymes such as lysozymes.
Use sonication - high frequency sound waves
How are proteins measured?
Lowry method - Folin reagent (Sodium tungstate molybdate and phosphate) mixed with copper sulphate solution and the protein. Gives a blue-purple colour which can be quantified by is absorbance at 660nm.
Bradford Method- used Coomassie Brilliant Blue dye which binds to protein- and then absorbs light at 595nm
What is important to remember about using the Lowry method?
- Partly dependent on tyrosine and tryptophan content of sample
- Tris, Hepes and EDTA can interfere with assay- so need in controls
What is important to remember about the Bradford method?
- Reagent quick to prepare
- Colour develops rapidly and is stable
- Not as sensitive as Lowry method and dependent on content of basic amino acids (arginine and lysine).
How are proteins purified?
low speed centrifugation to remove debris.
Digest DNA and RNA is DNA/RNA ases respectively
Salt fractionation with ammonium sulphate - as salt conc increases different proteins precipitate.
Centrifugation
Chromatography to separate the proteins
What different forms of chromatography can be used to separate proteins?
- Ion-exchange- based on charge
- Affinity- based on binding to a specific ligand
- Exclusion- based on size
What is exclusion chromatography?
Sephadex or Sepharose beads.
Beads have small hole in proteins
can either run around beads or in them (dependent on size)
Small proteins retarded, large ones rune around outside and washed out quickly.
Also for determining size of protein, concentration or desalting protein prep
How does a calibrate column work?
Run a mix of proteins of known molecular masses
See where elute and compare to your protein
What methods are used to determine the structure of a protein?
Gel filtration chromatography
Poly-acrylamide gel electrophoresis
Mass spectrometry
How does poly-acrylamide gel Electrophoresis work?
- Matrix made up by cross links between acrylamide and bis- acrylamide
- Limits movement of large proteins but allows smaller proteins through
- Vary percentage of gel depending on size of proteins wish to separate
- 8% for large proteins (180-60 kDa), 12% for small proteins (<60kDa)
- 4% “stacking gel” at the top with wells to load proteins into, concentrate at boundary between gels
What is the SDS-page method of gel electrophoresis?
Allows us to separate proteins only on size.
Cells containing proteins Cell lysis by detergents and sonication. Load proteins on to gel and apply current SDS Detergents bind to proteins Heat denaturation of proteins.
What is the advantage of using a native gel as opposed to SDS page?
Proteins are separated by size and charge of the proteins. Can look for enzymatic activity since the proteins are not denatured unlike SDS-page where the proteins are denatured.
What is the principle of mass spectrometry?
Knowing the speed and size of force applied onto an object enables us to calculate the mass of the object if we know the curve path it is deflected through. The less deflection, the heavier the object.
Therefore the smaller the object, the more it will be deflected from a sideways force.
How does mass spectrometry work?
Only needs picomole quantities of protein
• Stage 1: Ionization: The atom is ionised by knocking one or more electrons off to give a positive ion. This is true even for things which you would normally expect to form negative ions (chlorine, for example) or never form ions at all (argon, for example). Mass spectrometers always work with positive ions.
• Stage 2: Acceleration: The ions are accelerated so that they all have the same kinetic energy.
• Stage 3: Deflection: The ions are then deflected by a magnetic field according to their masses. The lighter they are, the more they are deflected. The amount of deflection also depends on the number of positive charges on the ion - in other words, on how many electrons were knocked off in the first stage. The more the ion is charged, the more it gets deflected.
• Stage 4: Detection: The beam of ions passing through the machine is detected electrically.
