Total Human PAI-1 ELISA Protocol

Reagents from Molecular Innovations
MA-31C9: Mouse anti-human PAI-1 (capture)
http://www.mol-innov.com/item/Mouse-monoclonal-to-human-PAI-1-MA-31C9

CPAI: Human PAI-1 (stable mutant)
http://www.mol-innov.com/item/Human-PAI-1-stable-mutant-form

ASHPAI-GF: Rabbit anti-human PAI-1 IgG Fraction
http://www.mol-innov.com/item/Rabbit-anti-human-PAI-1-IgG-fraction

GAR-HRP: Goat anti-rabbit IgG, HRP Labeled
http://www.mol-innov.com/item/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)
Pierce Superblock
ELISA Wash Buffer
TMB HRP Substrate
1M H2SO4

Protocol
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
50 ng/ml
20
10
5
2.5
1
0.5
0.2
0.1
0.05
0 (Blank)

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