Amino Acids, Proteins and Porphyrins
Same charged two proteins separation is done by
Purification of Proteins: -
Two major process: I — Chromatography (CG)
II— Electrophoresis (EP)
a. Usually consists of a mobile phase and a stationary phase.
b. Interactions between these two phase — include physio-chemical principle such as: -
Adsorption, partition, Ion exchange, molecular sieving and Affinity.
A. Partition CG -
a. Paper chromatography → Ascending and Descending
b. Thin Layer CG. (TLC)
c. Gas liquid CG (GLT)
B. Adsorption column C.G.
C. Ion — exchange chromatography: -
Ion-exchange-cations exchangers and Anion exchangers- are used
a. An anion exchanger (R+A-) exchange its Anion (A-) with another Anion (B-) is solution
R+ A- + B- ⇔ R+ B- +A-
b. A cation exchangers (H+R-) — exchanger with another cation (C+) in solution
H+ R- +C+⇔ C+ R- + H +
Now, clear from above that some charged are exchanged (separated)
Eg.of Ion exchange chromatography —
A. Polystyrene Resins (Anion exchange resin, Dowex 1; cation exchange resin, Dowex 50)
B. DEAE cellulose (= Diethyl Amino ethyl cellulose)
C. CM cellulose
F. Gel filtration C.G
G. Affinity C.G.
F. High performance Liquid C.G. (HPLC)
(II) Electrophoresis: The movements of charged particles (ions) in an electric field resulting in their migration towards the oppositely charged Electrode.
A. Zone Electrophoresis →eg
a. Electrophoresis →serum proteins are separated into five bands
b. Gel electrophoresis:-eg of gel commonly used in gel Electrophoresis are —
B. Poly acrylamide
C. Sodium dodecyl sulfate (SDS)
D. SDS -PAGE
B. Isoelectric focussing→at Isoelectric pH
C. Immuno electrophoresis