Reagents from Molecular Innovations
MA-31C9: Mouse anti-human PAI-1 (capture)
CPAI: Human PAI-1 (stable mutant)
ASHPAI-GF: Rabbit anti-human PAI-1 IgG Fraction
GAR-HRP: Goat anti-rabbit IgG, HRP Labeled
Other Commercial Reagents
10X TBS (1.0M Tris-HCl, 1.5M NaCl, pH 7.4)
1X TBS (0.1M Tris-HCl, 0.15M NaCl, pH 7.4)
TBS BSA (3% BSA in 1X TBS)
ELISA Wash Buffer
TMB HRP Substrate
1. Coat plate with PAI-1 capture monoclonal antibody at 5 ug/ml in TBS with 100 ul per well overnight.
2. Block with Superblock from Pierce or a similar ELISA plate blocking reagent according to manufacturer’s directions.
3. Make dilutions of human PAI-1 stable mutant in TBS BSA as shown in table below.
4. Apply 100 ul of each standard in duplicate along with unknown samples to wells. Shake at 300 rpm at room temperature for 30 minutes then wash 3x with wash buffer.
5. Detect with rabbit polyclonal to human PAI-1 IgG at 10 ug/ml in TBS BSA with 100 ul per well. Shake at 300 rpm at room temperature for 30 minutes then wash 3x with wash buffer.
6. Apply HRP conjugated secondary antibody to rabbit IgG at 1:10,000 in TBS BSA with 100 ul per well. Shake at 300 rpm at room temperature for 30 minutes then wash 3x with wash buffer.
7. Apply HRP substrate with 100 ul per well. Shake at 300 rpm at room temperature until color develops.
8. Quench reaction by adding 1M H2SO4 with 50 ul per well.
9. Determine Absorbance at 405 nm with microplate reader. Subtract Blank value from all other points. Generate standard curve and determine PAI-1 concentration of unknown samples.
Standard Curve Dilutions
#1 100 mM potassium phosphate, pH 7.4
150 mM KCl
1 mM EDTA
#2 100 mM tetrasodium pyrophosphate, pH 7.4
1 mM EDTA
#3 100 mM potassium phosphate, pH 7.4 (Suspension Buffer)
1 mM EDTA
1. Wash chopped tissue several times with ice cold saline solution.
2. Weigh tissue and add 4x volume of Buffer #1
3. Homogenize in Waring blender using two 45 second blends
4. Centrifuge mixture for 35 min at 9,000 g in a Sorvall centrifuge (GSA rotor; low speed centrifuge)
5. Discard pellet
6. Filter supernatant through 4 layers of cheesecloth
7. Spin filtered supernatant at 105,000 g for 75 minutes in a T35 rotor. (ultracentrifuge)
8. Suspend pellets in a minimal volume of Buffer #2 using a Potter-Elvehjem homogenizer.
9. Spin at 105,000 g for 75 minutes in a T35 rotor. (ultracentrifuge)
10. Suspend pellets in Buffer #3 using a Potter-Elvehjem homogenizer. Use ~1 mL of buffer / 2 grams of tissue.
11. Store in 1.0 mL aliquots in –70ºC.
This general protocol is is intended for use as a reference as a courtesy to our customers. Optimal concentrations, experimental conditions and experimental processes are to be determined by the individual user. No guarantee of performance using the above procedure is expressed or implied. Molecular Innovations does not currently perform homogenization of tissues in-house and can not provide further technical support for this application.
In most cases, collecting plasma in acidified citrate (acifidied citrate recipes) will preserve activity of PAI-1 or tPA in plasma. Published references have recommended the use of Biopool Stabilyte tubes for plasma collection. Unfortunately these tubes have been discontinued.
Tissue plasminogen activator activity is unstable in blood plasma. The activity half life ranges from 1 minutes to 4 hours depending on the level of PAI-1 activity. With other collection methods, i.e. anti-coagulants tri-sodium citrate or EDTA, it is impossible to prevent the fast inhibition of tPA by PAI-1. This results in underestimation of circulating levels of tPA activity. The Biopool® Stabilyte tube contains citrate anticoagulant at low pH (patented, ref. 4). The Biopool® Stabilyte tube immediately reduces the blood pH to about 5.9, inhibiting PAI 1/tPA complex formation and preserving the components of the fibrinolytic system. The components regain full activity after plasma neutralization.
Stabilyte tubes may be ordered from:
DiaPharma Group, Inc.
8948 Beckett Road
West Chester, Ohio 45069
Customer Service and Order Line: 1-800-526-5224
Technical Support and Product Information Line: 1-800-447-3846
General e-mail requests: firstname.lastname@example.org
Website URL: http://www.diapharma.com
The following anticoagulants are recommended for PAI-1, tPA and uPA activity ELISA kits.
Stabilyte sodium citrate (pH 4.3)
Dissolve 66.2 g sodium citrate dihydrate in 450 ml distilled water. Adjust the pH to 4.3 then adjust volume to 500 ml with water. Sterilize by filtration through 0.22 um filters. Store up to 2 weeks at 4C.
Acidified sodium citrate (pH 5.2)
Dissolve 37.3 g sodium citrate and 8 g citric acid in 450 ml distilled water. Adjust the pH to 5.2 with NaOH, then adjust volume to 500 ml with water. Sterilize by filtration through 0.22 um filters. Store up to 2 weeks at 4C.