Basic sad facts

1
Basic (sad) facts

• All proteins available in large amount and easy
  to purify have been extensively studied and are
  therefore not interesting
• Proteins are present in complex mixtures
• Proteins are fragile molecules that get easily
  modified and chopped up
• Interesting proteins are parts of complexes with
  other proteins and other molecules or are
  membranal
• Most genes are known
• Old purification methods are good but knowledge
  about them is not in the Web
• Cell walls and hard tissues are hard to break

2
The grand scheme of protein extraction and
purification
Extraction
clarification
precipitation
dialysis
Hydrophobic interaction (add salt)
affinity
ion exchange (remove salt)
Hydroxy hapatite and other minerals
Metal chelate
concentration
Gel permeation
Chromatofocusing
Reversed phase
3
Goal and amounts

• How much of the protein do we need?
• What is it going to be used for?
• How pure do we want it?
• Are there toxins in the sample?
• Identification and posttranslational modification
  analysis
• Crystallization
• Medicine

4
Disruption techniques

• Gentle
• Osmotic disruption
• Enzyme digestion
• Chemical disruption
• Hand homogenizer
• Mincing, grinding
• Moderate
• Blade homogenizer (Waring type, keep your
  fingers)
• Sand and alumina
• Freeze thaw or freeze and grind (frostbites)
• Vigorous
• French press (watch for bubbles
• Ultrasonication (watch for temp)
• Bead mill (watch for binding)
• Pressure cell (watch for explosions)

5
Ways to destroy proteins

• Heat (or cold and freezing)
• Proteolysis
• Organic solvents and detergents
• Chaotropic salts and chemicals
• Air
• Extreme pH
• Shear force
• Chemical modifications (including oxidation)
• Aggregation
• High salt or low salt

6
Good buffers?

• Buffer capacity
• Check the actual pH
• Check the temperature
• Check the pH after dilution and temperature
  change
• Make sure it does not interfere with the activity
  assay and the chromatography
• May prevent cross-linking
• Permeates membranes?
• Binds metals?
• Anion, cation or zwitterion?
• Organic or mineral?

7
Inhibiting proteases

• Cell and tissue disruption will always release
  proteases
• Purification will remove natural proteases
  inhibitors
• Exposed domains are always more sensitive
• Denaturation does not inhibit all proteases

8
Quantization of total protein

• Color reactions (Lowry)
• Absorbance (280 W,Y,F, 205 all aa)
• Dye binding (Coomassie, essentially irreversible)
• Amino acid analysis (the only accurate method)
• Accepted by authorities?

9
Detergents

• Ionic or non-ionic
• Denaturing or mild
• Pure or complex
• Cheap or expensive
• Mass spectrometry friendly?
• Critical micelle concentration (CMC)
• Can be removed?

10
Protease inhibitors

• Serine proteases (PMSF, TLCK,TPCK, nerve poisons,
  Soybean trypsin inhibitor)
• Cysteine protease (iodoaceteamide, DTT)
• Aspartic proteases (pepstatine)
• Amino and carboxyl peptidases (amastatine,
  bestatine)
• Metal proteases (EDTA)
• Serum protease inhibitors
• Carrier proteins
• Reversible and irreversible inhibitors
• Use of protease inhibitors for depletion of the
  proteases

11
additives

• Sucrose and glycerol to increase viscosity
• Metal chelators to prevent oxidation
• Sulfhydryl reagents to prevent formation of S-S
  bonds
• Ligands to protect active site of enzymes
• Carrier proteins, detergents and polymers to
  prevent aggregation and non-specific binding
• Salt to prevent salting-out or denaturation
• Bacteriostatic
• Low temperature

12
Extract them all together or pre-fractionate?

• Organelles are simple to fractionate in some
  cases
• Protein complexes can be spun down
• Salts and organic solvents will precipitate some
  proteins
• Precipitate fraction of the proteins (high salt
  or high organic solvent, pH close to pI)
• Separate by size
• Separate by phases dextran/PEG