Affinity Chromatography Media

CellufineTMPhosphate

For nucleic acid relate protein, dehydrogenase, phosphate relate protein, cation exchange chromatography

Cellufine Phosphate is an affinity media designed for concentration, purification of proteins and, enzymes such as nucleic acid related proteins. Base of media is spherical and rigid cellulose functionalized with Phosphate esters.

Partial Structure of Cellufine Phosphate
Figure1Partial Structure of Cellufine Phosphate
Characteristics
Support Matrix Cellulose
Ligand Phosphate ester
Ligand conc. 0.3 - 0.8meq/ml
Adsorption Capacity ≧ 20mg/ml-gel (lysozyme)
Calibration carves of Cellufine Phosphate and a conventional Cellulose phosphate.
Figure2Calibration carves of Cellufine Phosphate and a conventional Cellulose phosphate.

C.I.P. Stability test

Cellufine Phosphate is stable to cleaning in place (C.I.P) by alkali.

CIP condition : 0.2mol/L NaOH 3CV 0.05M phosphate buffer, pH7 5CV (repeat)
Figure3CIP condition : 0.2mol/L NaOH 3CV 0.05M phosphate buffer, pH7 5CV (repeat)
Use of Cellufine Phosphate in Rus A DZON purification
Figure4Use of Cellufine Phosphate in Rus A D70N purification

Chromatography

Column
1.6x10cm (20ml) packed with Cellufine Phosphate
Flow rate
3ml/min( 90cm/h )
Sample
7.5mg of RusA D70N obtained after Heparin-Sepharose chromatography
Gradient
200ml from 0.1 to 1.3M NaCl in 50mM tris-HCl pH 8.0

SDS-PAGE

Gel
Novex 4-12%BT gel used with MES-SDS running buffer (Invitrogen)
Key for gel
1 Cell free extract
2 Unbound material from Heparin-Sepharose column
3 RusA sample obtained from Heparin-Sepharose column
4-6 Fractions across the peak eluted from Cellufine Phosphate
7 RusA reference sample
8 Mark 12 MW standard (Invitrogen)

Ref.
Nucleic Acids Research, 2006, Vol. 00, No. 00 1–8
Rachel Macmaster, Svetlana Sedelnikova, Patrick J. Baker, Edward L. Bolt1,Robert G. Lloyd1 and John B. Rafferty
RusA Holliday junction resolvase: DNA complexstructure—insights into selectivity and specificity

This data was carried by courtesy of Dr. Svetlana Sedelnikovaof the Sheffield university.

Separation of mixed sample
Figure5Separation of mixed sample
Column Size
ID 1.1 cm – Height 10 cm
Flow rate
2 ml/min (126cm/h)
Buffer
0.01M acetate buffer, pH4.8
Elution
0 to 1 mol/L NaCl gradient

Development of the grade

Development of the grade according to demand of a customer (inquiry)

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