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Lect 5 B HIC
Lect 5 B HIC
Source of protein
Principle of HIC
Advantages of using HIC
Extraction What are the factors affecting HIC
Example: Isolating linamarase with
HIC
Separation
Purity &
characterization
HIC
Hydrophobic interaction chromatography
Source of protein Principle
Separation of substances is based on
their varying strength of interaction
Extraction with hydrophobic groups attached to
an uncharged gel matrix
HIC
Hydrophobic interaction chromatography
General concept
Salt-promoted adsorption… Porath(1986)
L H Soluble protein
L H Soluble protein
In medium containing
ammonium sulfate
L
H Soluble protein
L H Soluble protein
HIC
What is the experimental set up like?
Fraction
Column
collector
Recorder
Buffer
UV monitor
HIC
Hydrophobic Interaction Chromatography
Source of protein
Principle of HIC
; Advantages of using HIC
Extraction What are the factors affecting HIC
Example: Isolating linamarase with
HIC
Separation
Purity &
characterization
HIC
Advantages of HIC
HIC
Hydrophobic Interaction Chromatography
Source of protein
Principle of HIC
Advantages of using HIC
Extraction ; What are the factors affecting HIC
Example: Isolating linamarase with
HIC
Separation
Purity &
characterization
HIC
Factors affecting HIC
HIC
Factors affecting HIC
HIC
Factors affecting HIC
Degree of substitution
HIC
Factors affecting HIC
HIC
Factors affecting HIC
HIC
Factors affecting HIC: pH
HIC
Factors affecting HIC: temperature
HIC
Factors affecting HIC: additives
HIC
Hydrophobic Interaction Chromatography
Source of protein
Principle of HIC
Advantages of using HIC
Extraction What are the factors affecting HIC
; Example: Isolating linamarase with
HIC
Separation
Purity &
characterization
HIC
Linamarase preparation using HIC:
Novelty: instead of ammonium sulfate precipitating the
protein, use salt to precipitate undesired protein
Leaf
Bind filtrate to
column; was 3 ml Phenyl
with 6 ml 0,6M Sepharose
Amm sulfate;
2 g tissue in 10 discard eluate;
ml 100 mM Na wash column
citrate pH 6; add with 6 ml
0.1 g PVP, then water; collect
homogenize and eluate
followed by 12
ml 2M Amm
sulfate
Peel (root) Final enzyme preparation
HIC
(90 min)
Crude enzyme extract
Salt precipitation
Dialysis step
Hydrophobic
Chromatofocusing
Interaction
Chromatography
HIC
Summary
HIC