yes

Fibrinogen – plate coating

Protocol for coating wells with fibrinogen for cell adhesion

1. Dilute fibrinogen to 10 ug/ml in 0.05 M Na Phosphate, 0.15 M NaCl, pH 7.4 (PBS).
2. Sterile filter coating solution.
3. Coat wells and incubate at 4C overnight.
4. Wash wells three times with PBS.
5. Plate cells.

Fibronectin – plate coating

Protocol for coating wells with fibronectin for cell adhesion

1. Dilute fibronectin to 10 ug/ml in 0.05 M Na Phosphate, 0.15 M NaCl, pH 7.4 (PBS).
2. Coat wells and incubate at 4C overnight.
3. Wash wells three times with PBS.
4. Plate cells.

Vitronectin – plate coating

Protocol for coating wells with vitronectin for cell adhesion

1. Dilute vitronectin to 10 ug/ml in 0.05 M Na Phosphate, 0.15 M NaCl, pH 7.4 (PBS).
2. Coat wells and incubate at 4C overnight.
3. Wash wells three times with PBS.
4. Plate cells.

Plasmin Activity

1 mg of plasmin contains 24 CU or 28 CTA-U.

Our purified plasmins (HPLM, BPLM, MPLM, RPLM, RbPLM) are 100% active because they are purified on immobilized SBTI, which only binds functionally active plasmin.

The amount of plasminogen in 1 ml of normal plasma (approximately 0.2 mg) corresponds to 3.8 CU or 27.74 nkat S-2251 when activated by streptokinase (1). In 1983 Friberger (2) reported that 1 µg of human plasmin corresponds to 0.20 nkat S-2251, or to 0.024 CU or to 0.028 CTA-U. This specific activity conversion factor can also be applied to plasmin from other species. In 1975 a preparation of partially purified plasmin in 50% glycerol was established as an international reference preparation (3). The World Health Organization defined the International Unit of Plasmin as the activity contained in 0.125 ml of the International Reference Preparation of Plasmin (4).

Abbreviations:
nkat S-2251 – 1 nkat = 1 x 10-9 mol of product released per second from plasmin substrate S-2251
CU – Casein Units
CTA-U – Committee on Thrombolytic Agents (CTA) Units

References:
1. Friberger P and Knos M. Plasminogen activation in normal plasma. In: Chromogenic peptide substrates, Eds, Scully MF and Kakkar VV, Churchill Livingstone, 128-140 (1979).
2. Friberger P et al. Synthetic peptide substrate assays and fibrinolysis and their application on automates. In: Seminars in Thrombosis and Haemostasis, Vol. 9, 281-300 (1983).
3. Kirkwood TB et al. A standard for human plasmin. Thromb Diath Haemorrh 34, 20-30 (1975).
4. 28th Report, WHO Expert Committee on Biological Standardization. WHO Tech Rep Ser No 610; 1977, pp 13.

How are the clotting assays performed on your coagulation factors?

Standard Curve:
An eight point standard curve is generated using the MLA 1400C. NPP Plasma (made and characterized in house) dilution is started at a 1:5 and subsequently serial diluted. To this dilution, an equal volume of FVII deficient plasma is added (1:1). The dilution is incubated at 37ºC for 106 seconds. Clotting is initiated by addition of 2 parts PT reagent [Hemoliance RecombiPlasTin; Lexington, MA]. A new standard curve was generated for each testing day.

Test Samples:
The purified samples are prepared by making a dilutions into deficient plasma. These samples are then serially diluted in Assay Buffer by the MLA 1400C from a 5-fold dilution for a total of three dilutions. The MLA 1400C compares the clot times to the standard curve and expresses the result as a percentage on the printout.

Urokinase (uPA) Assays

To test the uPA activity of your sample, we recommend the following protocol for a 1.0 ml microcuvette:
1. Add 900 ul of uPA chromogenic substrate at 100 uM in TBS or PBS
2. Add 5 ul of uPA sample to cuvette and mix well
3. Run kinetic protocol at 405 nm for 60 seconds

To determine the percentage of your uPA that is still active, we recommend incubating 0.5 – 1.0 ug of uPA with a five fold molar excess of active PAI-1 in a neutral pH buffer, then running the sample on SDS-PAGE to check amount of uPA-PAI-1 complex that is formed. If the uPA was kept in the provided storage buffer (0.1 M Acetate, 0.1 M NaCl, 1 mM EDTA, pH 5.0) and frozen at -20 C or colder, there should be very little loss of activity.

For cleavage of proteins, we recommend adding a catalytic amount of uPA (usually a 1:100 molar ratio of uPA to target protein) and incubating in a neutral pH buffer. The reaction will proceed more slowly at room temperature than at 37 C. You should run pilot assays at a very small scale taking samples for SDS-PAGE at time points to determine the ideal time and temperature needed for complete cleavage without excessive non-specific degradation.

PAI-1 Inhibition Kinetic Assay with tPA

PAI-1 Inhibition of tPA
1. tPA Control Rate – Incubate 5 ug of human or mouse tPA in a total volume of 100 ul TBS for 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate at 10 mM (purchased from Centerchem), mix and take kinetic absorbance at 405 nm for 1 minute.
2. PAI-1 Inhibition Control Rate – Incubate 5 ug of tPA with 2 ug of fully active control PAI-1 in a total volume of 100 ul TBS for 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate, mix and take kinetic absorbance at 405 nm for 1 minute.
If the PAI-1 is 100% active, it should inhibit about 65% of the active tPA. The rate should be decreased 65% compared to the tPA Control Rate.
3. PAI-1 Sample Rate – Incubate 5 ug of tPA with 2 ug of sample PAI-1 in a total volume of 100 ul TBS for 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate, mix and take kinetic absorbance at 405 nm for 1 minute.
Compare the sample rate to the PAI-1 Inhibition Control Rate and the tPA Control Rate. If the PAI-1 in your sample has gone partly latent, it will inhibit less tPA than the 100% active PAI-1, so the rate will be greater than the PAI-1 Inhibition Control Rate. If the PAI-1 is completely latent, the rate will be equal to or greater than the tPA Control Rate. You can estimate the percent activity remaining in the sample by taking a ratio of the rates.

PAI-1 Inhibitory Antibody
Perform 1. tPA Control Rate and 2. PAI-1 Inhibition Control Rate as above.
3. Antibody Control Rate – Incubate 2 ug control PAI-1 with 3 ug of control inhibitory antibody in a total volume of 75 ul TBS for 5 minutes. Add 5 ug of tPA, increase the total reaction volume to 100 ul with TBS and incubate an additional 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate, mix and take kinetic absorbance at 405 nm for 1 minute.
Compare this rate to the PAI-1 Inhibition Control Rate and the tPA Control Rate. The antibody should inhibit about 80% of the PAI-1, which will only inhibit approximately 13% of the tPA. The rate should be close to the tPA Control Rate.
4. Antibody Sample Rate – Incubate 2 ug control PAI-1 with 3 ug of sample inhibitory antibody in a total volume of 75 ul TBS for 5 minutes. Add 5 ug of tPA, increase the total reaction volume to 100 ul with TBS and incubate an additional 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate, mix and take kinetic absorbance at 405 nm for 1 minute.
If the antibody has lost its inhibitory activity, the rate will be closer to the PAI-1 Inhibition Control Rate than the Antibody Control Rate.

Fibrin – plate coating

1. Dilute fibrinogen to 1.75 uM in 0.05 M Na Phosphate, 0.15 M NaCl, pH 7.4 (PBS).
2. Add 80 ul fibrinogen to each well.
3. Dilute thrombin to 7.5 units/ml in PBS.
4. Add 20 ul thrombin to each well.
5. Cover lightly with kimwipe and incubate at room temperature overnight. The fluid will evaporate, leaving the fibrin clot stuck to the plate.
6. Wash wells three times with PBS.

Plasminogen Activation to Plasmin by uPA

To determine complete activation of plasminogen to plasmin with uPA, follow this protocol:

1. Prepare a 10% Tris-Glycine SDS-PAGE protein gel, or similar gel capable of resolving 50-100 kDa.
2. Prepare three tubes with 10ul of TBS, 1ug of HGPG, and 0.1ug of uPA.
3. Create three time points by adding 10ul of reducing sample loading buffer to tubes at 1min, 10min, and 30min.
4. Prepare one tube with 10ul of TBS, 1ug of HGPG, and 10ul of reducing sample loading buffer to create a negative control.
5. Boil all tubes for 1min.
6. Run all samples on SDS-PAGE and Coomassie stain gel.
7. The negative control will appear as a single band at 92 kDa.
8. The time points will appear as multiple bands starting at 85 kDa.
9. Conversion to plasmin is indicated by disappearance of the band at 92 kDa.

Prorenin activity assay

Human prorenin is activated by controlled digestion until the conversion is complete as evidenced by a shift in molecular weight by SDS PAGE. We do not routinely measure enzymatic activity on each lot but this can be performed using the fluorescent peptide substrate (Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(dabcyl)-Arg) from Molecular Probes (Cat # R-2931). Activity is detected in a microplate format using a SpectroMax Gemini microplate reader. We have done this before and observe a dramatic increase in substarte hydrolysis subsequent to prorenin activation. Prorenin itself has only 1-5% of the enzymatic activity of the renin produced after activation.

Specific activity is determined to be 100 or greater pmoles of substrate hydrolyzed/min/μg.