Facile purification of fibrinogen fragments using a computer-based model with general applicability to the generation of salt gradients.
We and others have recently shown that specific fragments of cross-linked fibrin affect cell behavior. In order to develop a facile method for the preparative scale purification of fibrin fragment D dimer, a simple gradient generating system for conventional chromatography was developed and validated, and methods of fibrin fragment D dimer purification were compared. The experimentally measured salt concentration/time relationship fell directly on the model-predicted line. Model-predicted changes in the reservoir volume and/or salt concentration in the limit buffer affected both the initial slope and the shape of the concentration/time relationship. This gradient generation method was used to separate the D domains of fibrin(ogen) from the amino terminal region E domain using anion-exchange chromatography. While the predicted salt gradient was achieved, a salt-dependent separation was found to be less optimal than that of a pH-dependent separation, as validated by Coomassie-stained SDS-PAGE and by immunoblotting. In conclusion, a facile, user-friendly, computer-based method to predict and generate salt gradients was written and validated by direct experimentation. While fibrinogen fragment purification was acceptable using this system, both separation and yields of fibrinogen and fibrin fragments were superior using a pH-based separation technique.
Olman
M A
MA
Department of Medicine in the Division of Pulmonary and Critical Care Medicine, University of Alabama, Birmingham, Alabama, 35294, USA.
Williams
W F
WF
Strickland
J H
JH
Hagood
J S
JS
Simmons
W L
WL
Rivera
K E
KE
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D005338:Fibrin Fibrinogen Degradation Products / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D012492:Salts; D012984:Software
D005338:Fibrin Fibrinogen Degradation Products; D012492:Salts; C036309:fibrin fragment D; C072054:fibrin fragment E-2; C011468:fibrinmonomer