Protein Purification

Protein Purification

1. Techniques of protein purification

   • Aims and objectives
     • Determine molecular mass and subunit composition using gel filtration
       • of oligomeric proteins
         • A molecular complex consisting of a few monomer units
           • Non-covalent bonding of few macromolecules
   • SDS-PAGE
     • Analyse extent of purity
       • determine molecular mass
     • Monitor efficiency of purification
       • Find yield and purification factor at each step
       • Determine increase of specific activity
         • As a result of purification procedure
     • Maths
       • Find relationship between logMW and distance travelled of known proteins to form a calibration curve
         • Look at distance travelled of haemoglobin (SDS denatured so can detect subunits)
       • Haemoglobin
         • Protein = 60kD
         • Subunit = 15.1kD
         • Made of 4 subunits
   • Seperating a mixture of proteins
     • Beta galactosidase
       • dyed
     • Cytochrome C
       • bound heme group
         • Used to calculate mass of Cytochrome C
     • B chain of insulin
       • modification by dinitrophenol
   • Packing column
     • Resin mixed with saline buffer and poured, avoiding formation of bubbles
       • Saline runs out and resin settles
         • Topped with resin and saline buffer added to prevent the column from drying out
           • Washed through a few times with pH suitable buffer
     • Use calibration curve on log graph to find molecular mass of cytochrome C

2. Aims and Objectives

   • determine quarternary structure.
     • Mr determined will be used in SDS-PAGE to find the number of subunits and quarternary state
   • Dithionite (reducing agent) enters all the beads so moves through the column slowly
     • haemoglobin enters 20% of the beads, when it catches up with dithionite, dithione removes the oxygen so the haemoglobin turns purple.
       • once it passes, it reoxidises and heamoglobin and forms orange

3. Analysis session 1

   • Specific activity (units mg-1)
     • number of units of enzyme activity per mg of protein
     • Specific activity = enzyme acitivity (units/ml) / protein concentration (mg/ml)
   • Percentage yield
     • (total activity after / total activity before) x 100
   • Total activity
     • enzyme activity (units/ml) x number of ml in sample
   • Purification factor
     • specific activity after / specific activity before

4. BSA calibration curve to determine protein concentration

   • Pools
     • Pool 1
       • mixture
     • Pool 2
       • no binding to Cibachrom Blue
     • Pool 3
       • non-specifically bound + salt
     • Pool 4
       • resin + glutamic acid
     • Measured to find success of purification
       • Compare proteins from pools 2, 3 and 4 to pool 1.
   • The concentrations of above measure to find success of purification
     • Compare proteins from pools 2, 3 and 4 to pool 1 to find a percentage

5. Glutamate dehydrogenase (GSH) catalysis

   • glutamic acid + NAD+ --> alpha ketoglutarate + NADH+ + H+
     • 1 mol glutamic acid = 1 mol NADH+
     • Enzyme activity and specific activity calculated