Since the ocriplasmin and their variants expressed in Pichia were in the inactive zymogen forms, it was necessary to be activated by the use of urokinase plasminogen activator (Sigma-Aldrich, U.S.A) to convert the purified ocriplasminogen variants into the corresponding active forms. We used different enzyme concentrations and incubation times to achieve the optimal time and concentration of urokinase enzyme for the activation of ocriplasmin. Solutions of the ocriplasminogen variants with concentrations of 5–20 mM were incubated at 37 °C for 10, 30 and, 60 min, in the presence of the urokinase at an ocriplasminogen to urokinase ratio of 100/2 and 100/5. After completing the phase of activation, SDS-PAGE was performed to evaluate the conversion of ocriplasminogen to the active form.
Urokinase plasminogen activator
Manufactured by Merck Group
Sourced in United States, Germany
Urokinase plasminogen activator (uPA) is a laboratory reagent used in research applications. It is a serine protease that activates the conversion of plasminogen to plasmin, which is involved in various physiological and pathological processes. uPA plays a role in extracellular matrix degradation, cell migration, and tissue remodeling. It can be used in research studies related to these biological functions.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated immunoglobulins, by Agilent Technologies (3 mentions)
Human glu plasminogen, by Prolytix (3 mentions)
Fibrinogen, by Merck Group (2 mentions)
D val leu lys p nitroanilide dihydrochloride, by Merck Group (2 mentions)
S 2251, by Merck Group (2 mentions)
Plasminogen, by Merck Group (1 mentions)
Mouse anti his antiserum, by GE Healthcare (1 mentions)
Anti c5b 9 antibody, by Quidel (1 mentions)
Factor h, by Complement Technology (1 mentions)
Factor b, by Complement Technology (1 mentions)
5 protocols using urokinase plasminogen activator
1
Activation of Recombinant Ocriplasmin
2
Embryo Culture Optimization Using IGF2, uPA, and Plasminogen
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The media used for embryo culture was EmbryoAssist (stage 1) and BlastAssist (stage 2) sequential media system (MediCult A/s, now ORIGI A/s Denmark). All embryos were cultured in EmbryoAssist media overnight for 20 h and assessed for development to the 2-cell stage.
The 2-cell embryos were pooled into a single dish and then randomly allocated into one of four treatment groups: (1) control, which were cultured in EmbryoAssist media alone; (2) IGF2, which were cultured in media containing 12.5 nM IGF2 (GroPep, Thebarton, SA, Australia); (3) U + P, which were cultured in media containing 10 µg/mL urokinase plasminogen activator (uPA; Sigma-Aldrich) and 5 µg/mL plasminogen (Sigma-Aldrich); or (4) IGF2 + U + P treatments, which were cultured in media containing a combination of 12.5 nM IGF2, 10 µg/mL urokinase plasminogen activator and 5 µg/mL plasminogen.
Embryos were washed and then cultured in this media for a further 23 h (Fig. 1). Cleavage stage embryos were then transferred to BlastAssist media with appropriate treatment for further 48 h. All embryo culture dishes were equilibrated to appropriate temperature and gas mix for at least 4 h before culture.
The 2-cell embryos were pooled into a single dish and then randomly allocated into one of four treatment groups: (1) control, which were cultured in EmbryoAssist media alone; (2) IGF2, which were cultured in media containing 12.5 nM IGF2 (GroPep, Thebarton, SA, Australia); (3) U + P, which were cultured in media containing 10 µg/mL urokinase plasminogen activator (uPA; Sigma-Aldrich) and 5 µg/mL plasminogen (Sigma-Aldrich); or (4) IGF2 + U + P treatments, which were cultured in media containing a combination of 12.5 nM IGF2, 10 µg/mL urokinase plasminogen activator and 5 µg/mL plasminogen.
Embryos were washed and then cultured in this media for a further 23 h (Fig. 1). Cleavage stage embryos were then transferred to BlastAssist media with appropriate treatment for further 48 h. All embryo culture dishes were equilibrated to appropriate temperature and gas mix for at least 4 h before culture.
3
Plasmin Activation Assay Protocol
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Human glu-plasminogen was obtained from Haematologic Technologies (Essex Junction, VT, USA). Plasminogen was activated to plasmin using urokinase plasminogen activator (uPA) from Merck Millipore, Darmstadt, Germany. Both the chromogenic substrate S-2251 (D-Val-Leu-Lys p-nitroanilide dihydrochloride) and fibrinogen were purchased from Sigma-Aldrich (Steinheim, Germany). Goat anti-fibrinogen antiserum was purchased from Acris Antibodies (Herford, Germany), mouse anti-His antiserum was obtained from GE Healthcare (Munich, Germany). Horseradish peroxidase (HRP)-conjugated immunoglobulins were purchased from Dako (Hamburg, Germany).
4
Plasmin Activation and Measurement
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Human glu-plasminogen was obtained from Haematologic Technologies (Essex Junction, VT, USA). Plasminogen was activated to plasmin using urokinase plasminogen activator (uPA) from Merck Millipore, Darmstadt, Germany. Both the chromogenic substrate S-2251 (D-Val-Leu-Lys p-nitroanilide dihydrochloride) and fibrinogen were purchased from Sigma-Aldrich (Steinheim, Germany). Purified C3b was obtained from Complement Technology, Tyler, TX, USA. A. baumannii Tuf was detected using a polyclonal rabbit antiserum raised against Streptococcus pneumoniae Tuf [29 (link)]. C3 and fibrinogen polyclonal antisera were purchased from Acris Antibodies (Herford, Germany). The monoclonal hexahistidine antibody was obtained from GE Healthcare (Munich, Germany). Horseradish peroxidase (HRP)-conjugated immunoglobulins were purchased from Dako (Hamburg, Germany) and Alexa Fluor 488-conjugated anti-rabbit immunoglobulins from Life Technologies (Darmstadt, Germany).
5
Plasminogen Activation and Complement Regulation
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Human serum (NHS) was collected from healthy blood donors as described previously50 (link). Human glu-plasminogen was purchased from Haematologic Technologies (Essex Junction, VT, USA) and urokinase plasminogen activator (uPA) (Merck, Darmstadt, Germany) were used for the activation of plasminogen to plasmin. The chromogenic substrate S-2251 (D-Val-Leu-Lys p-nitroanilide dihydrochloride) were from Sigma-Aldrich (Steinheim, Germany). Factor H, Factor B, C3b, and C5 were purchased from Complement Technology (Tyler, TX, USA). Polyclonal anti-plasminogen antibody was purchased from Acris Antibodies (Herford, Germany), and the monoclonal anti-plasminogen antibody (clone 10-V-1) was from Calbiochem, Merck, Darmstadt, Germany). The polyclonal anti-FH and anti-C3 antibody were obtained from Merck Biosciences (Bad Soden, Germany) and the polyclonal anti-C5, anti-Factor B antibody as well as the neoepitope-specific monoclonal anti-C5b-9 antibody was from Quidel (San Diego, CA, USA). The mouse anti-His antiserum was obtained from Novagen (Merck Darmstadt, Germany) and Qiagen (Hilden, Germany) and the horseradish peroxidase (HRP)-conjugated immunoglobulins were purchased from Dako (Hamburg, Germany).
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