Migration of the activation peptide of thrombin-activatable fibrinolysis inhibitor (TAFI) during SDS-polyacrylamide gel electrophoresis.
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which upon activation is capable of delaying fibrinolysis. We investigated the migration and detection of the activation peptide of TAFI during SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Purified TAFI before and after activation by thrombin/thrombomodulin was electrophoresed on 4-20% polyacrylamide gels and stained with Coomassie blue as well as Western blotting. Before activation, Coomassie blue staining resulted in one main band of TAFI. After activation, a sharp band corresponding to TAFIa was observed. No distinct activation peptide was detected, in agreement with the literature. Western blotting using a polyclonal anti-TAFI antibody, on the other hand, showed one additional broad band with an Mr of about 33 000 after TAFI activation. N-terminal sequence analysis confirmed that this band represented the activation peptide of TAFI. In addition, we tested the reactivity of two anti-TAFI monoclonal antibodies (MA-T3D8 and MA-T18A8) towards TAFI before and after activation by Western blotting. Both monoclonal antibodies recognized TAFI. After activation of TAFI, MA-T3D8 reacted with TAFIa, while MA-T18A8 reacted with the activation peptide. We identify the 33 000 band as the activation peptide of TAFI and exemplify the use of this information for the characterization of monoclonal antibodies against TAFI.
Guimarães
A H C
AH
Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands.
Barrett-Bergshoeff
M M
MM
Gils
A
A
Declerck
P J
PJ
Rijken
D C
DC
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D025901:Carboxypeptidase B2 / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D025901:Carboxypeptidase B2