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chromatography
(AEC)
Matrix or Resin
Matrix Is Support
Covalently attached functional group (charged)
Matrix, polymeric, porous beads
Cellulose
Sepharose: is a tradename for a crosslinked, beadedform of a polysaccharide polymer material extracted
from seaweed. It is crosslinked through lysine side
chains.
Sephacel: bead form of cellulose
Sephadex: crosslinked dextran/bead form of dextran
Packed (poured) into the column as a slurry
IEC Groups
Anion exchange chromatography beads
(matrix/resins)
Carboxymethyl (CM)
(Sulfate derivative)
Quaternary amine
Diethylaminoethyl (DEAE)
Cellulose Matrix
Aminoethyl-Cellulose
Exchange Type
Weak
Weak
Polyethylenimine (PEI)-Cellulose
Weak
Diethyl-[2-hydroxypropyl]-aminoehtyl
Strong
(DAE)-Cellulose
Triethylaminoethyl (TEAE)-Cellulose Weak
Diethylaminoethyl (DEAE)-Sephacel Weak
Exchange Type
Strong
Strong
Intermediate
Strong
Strong
Sephadex Matrix
Diethylaminoethyl (DEAE)-Sephadex
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)Sephadex
Exchange Type
Weak
Sepharose Matrix
Diethylaminoethyl (DEAE)-Sepharose CL-6B
Diethylaminoethyl (DEAE)-Sepharose (fast flow)
Quaternary amine (Q)-Sepharose (fast flow)
Strong
Exchange Type
Weak
Weak
Strong
Steps of
Anion Exchange chromatography (AEC)
I. Set up column; other equipment
II. Choose buffer (pH important, above protein pI for AEC)
III.Prepare resin; pour column; equilibrate resin with buffer
IV.Load sample onto column --adsorption
V. Wash unbound proteins off the column
VI. UV spectrophotometer= OD 280 nm
VII.Desorption; elution (step gradient/continous gradient)
(Collect fractions of 1ml or 5 ml or 10ml using fraction
collector)
VIII.End desorption; regenerate column (optional)
Chromatogram
Charge On Proteins
Overall net charge on a protein is
determined by the R groups of its
amino acids
Some amino acids have R group with
positive charge, others negative
charge, others no charge
Or
Two protein both negatively
charged will bind anion exchange
matrix !!!!!!!!!!
Can both be separated?
pH Of Environment
Is Crucial
pI Is Important
First, pI for a protein of interest
determines whether use cation or
an anion resin
pI
Also, usually dont want to use a pH
where the protein has no charge at
all because it will never stick to
the beads
So, for example, pI is 6.2, may want
to use a buffer at 7.2.
IEC Principles
Order of elution: +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution / desorption of
proteins bound to anion exchange
With Salt Gradient:
Counter anion Chloride ions (NaCl)
Increasing concentration
First least negatively charged proteins will elute
Followed by more negaitvely charged proteins
OR
With pH gradient
Lower the pH slowly
Proteins will slowly become less negative and
than positive as pH will reach below its pI
Practice
Next lecture
Cation exchange
chromatpgraphy (CEC)