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4 protocols using horseradish peroxidase hrp conjugated immunoglobulins

1

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).
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2

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).
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3

Immunoblotting Analysis of TLR-Induced Signaling

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Cells were treated with TLR‐agonists and siRNA as indicated in the figure legends. Then the cells were washed twice in PBS before they were lysed in lysis buffer (50 mM Tris–HCl, 1% NP40, 150 mM NaCl, 10% glycerol, 1 mM Na3VO4, 50 mM NaF and Complete protease inhibitor [Roche Diagnostics, Mannheim, Germany]). The samples were denatured in 1× NuPage LDS sample buffer supplemented with 25 mM DTT for 10 min at 70°C before they were separated on 10% Bis‐Tris polyacrylamide gel and transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen, Camarillo, California). The membrane was blocked using 5% bovine serum albumin (Sigma–Aldrich, St. Louis, MO) in Tris‐buffered saline with 0,1% Tween. TLR3‐ and pro IL‐1β antibodies (Cell Signaling Technology, Beverly, Missouri) were used for protein detection. Monoclonal anti‐GAPDH (Abcam, Cambridge, United Kingdom) was used as loading control. For visualization the blots were incubated with horseradish peroxidase (HRP) conjugated immunoglobulin's (DAKO, Glostrup, Denmark) and developed with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, Illinois). Images were obtained with LI‐COR Odyssey Fc and analyzed using Image Studio Software (LI‐COR, Lincoln, Nebraska).
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4

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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