C3 S3&4

lab method used to iso, purify, and characterize proteins

“salting out“ may be used in initial purification method

reduces solubility of proteins in soln. of high ionic strength

not a way to iso a specific protein

• used to iso a group, and to remove a group of proteins

MWCO

molecular weight cut off

dialysis

commonly used to remove small molecules and ions from protein soln.

used to change buffer conditions

(smaller pore = slower exchange)

Chromatographic methods

are separation methods 

have stationary phase and mobile phase

separate components of mixtures based on diff. in affinites for the phases

• charges

• size

• binding affinity

ion exchange

separation based on differences in net charge of mixtures components

• column material has:

  • covalently bound cation → (anion exchanger) OR

  • covalently bound anion → (cation exchanger)

When running a columm, what happens

1. condition the column

2. load the protein sample

3. wash the column

4. elute the desired protein → pH or salt changes

molecular sieve chromatography

separation based on size (also called: gel permeation, size exclusion)

column resin beads have pores:

• smaller the molecules → more time they spend there. slower they move down the column

• bigger the molecules → less time it spends in there, quicker it moves down

Affinity chromatography

based on specific interactions between a column-bound ligand and protein of interest

“his-tag, antibody, protein”

1. protein of interest is bound to column

   1. afterwards the unwanted proteins elute from column, POI is eluted by adding free ligand to mobile solvent

electrophoretic methods 

“more for analysis”

are separation methods 

separate the components of mixtures based on their differences in mobility through an electric field 

• + cations will move towards the cathode (- electrode)

• - anions move toward anode (+ electrode)

• net zero charges don’t migrate

  • rate of migration depends on charge & mass ratio 

examples of electrophoretic methods

agarose gel electrophoresis

polyacrylamide gel electro PAGE [used for smaller sized DNA proteins]

• SDS page

• isoelectric focusing

SDS page

sodium dodeccyl sulfate polyacrylamide gel electrophoresis

• denatures all proteins in mixture, encapsulates them

• gives them all negatively charged surfaces

• all proteins will migrate towards the anode

• separate based on their molecular wt diff.

  • smaller they are, easier for it to move through gel maxtrix

isoelectric focusing

proteins separated by diff. in PI

2D electrophoresis

IEF + SDS page

1st IEF - sep. by PI

2nd SDS - sep. by Size

using electrophoresis to determine if the protein is oo

oligomeric

means

more then one side chain/sub-units