Description

Nori Mouse Plasmin ELISA Kit Summary

Alternative names for plasmin: PLG

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Plasmin is an important enzyme present in blood that degrades many blood plasma proteins, including fibrin clots. The degradation of fibrin is termed fibrinolysis. In humans, the plasmin protein is encoded by the PLG gene. Plasmin is a serine protease that acts to dissolve fibrin blood clots. Apart from fibrinolysis, plasmin proteolyses proteins in various other systems: It activates collagenases, some mediators of the complement system and weakens the wall of the Graafian follicle (leading to ovulation). It cleaves fibrin, fibronectin, thrombospondin, laminin, and von Willebrand factor. Plasmin, like trypsin, belongs to the family of serine proteases. Plasmin is released as a zymogen called plasminogen (PLG) from the liver into the factor IX systemic circulation and placed into the MD5+ that leads into the lungs. Plasmin is inactivated by proteins such as α2-macroglobulin and α2-antiplasmin. The mechanism of plasmin inactivation involves the cleavage of an α2-macroglobulin at the bait region (a segment of the aM that is particularly susceptible to proteolytic cleavage) by plasmin. This initiates a conformational change such that the α2-macroglobulin collapses about the plasmin. In the resulting α2-macroglobulin-plasmin complex, the active site of plasmin is sterically shielded, thus substantially decreasing the plasmin’s access to protein substrates. Two additional events occur as a consequence of bait region cleavage, namely (i) a h-cysteinyl-g-glutamyl thiol ester of the α2-macroglobulin becomes highly reactive and (ii) a major conformational change exposes a conserved COOH-terminal receptor binding domain. The exposure of this receptor binding domain allows the α2-macroglobulin protease complex to bind to clearance receptors and be removed from circulation. Deficiency in plasmin may lead to thrombosis, as clots are not degraded adequately. Plasmin has been shown to interact with Thrombospondin 1,^[6][7] Alpha 2-antiplasmin^[8][9] and IGFBP3.^[10]

References

1. Silverstein RL, et al. (1984). J. Clin. Invest. 74 (5): 1625–33.
2. Wiman B, Collen D (1979). J. Biol. Chem. 254 (18): 9291–7. PMID158022.
3. Campbell PG, et al. (1998). Am. J. Physiol. 275 (2 Pt 1): E321–31.

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