Universal Protease Activity ELISA

$900.00

The Universal Protease Activity ELISA Assay Kit measures activity of proteases like matrix metalloproteases (MMPs), A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS), especially High temperature requirement factor A (HtrA). It consists of two modules: Protease module and the ELISA module. In this assay, a biotinylated ß-Casein coupled beads is first digested with protease(s)/HtrA1. The proteolytic cleavage of the substrate releases a biotinylated ß-Casein fragment, without beads, that will bind on streptavidin-coated plate and can be further quantified with one specific monoclonal antiß-Casein antibodies and with a Peroxidase-labeled antibody The Universal Protease Activity ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: 30 516 103 Categories: , ,

Universal Protease Activity ELISA

The Universal Protease Activity ELISA is For Research Use Only

Size: 1×96 wells
Sensitivity: 0.312 nM HtrA1
Dynamic Range: 0.039 – 2.5 nM
Incubation Time: 2.25 hours
Sample Type: Serum, tissue, cell culture supernatants
Sample Size: 10 µL
Controls Included


Assay Background

Proteases play important roles in the control of multiple biological processes in all living organisms. They regulate the activities of many proteins, modulate protein-protein interactions, create new bioactive molecules, contribute to the processing of cellular information and generate, transduce and amplify molecular signals. As a direct result of these multiple actions, proteases influence DNA replication and transcription, cell proliferation and differentiation, tissue morphogenesis and remodelling, heat shock and unfolded protein responses, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, haemostasis, blood coagulation, inflammation, immunity, autophagy, senescence, necrosis and apoptosis. Consistent with these essential roles of proteases in cell behaviour, survival and death of all organisms, alterations in proteolytic systems underlie multiple pathological conditions such as cancer, neurodegenerative disorders, inflammatory and cardiovascular diseases. The Universal Protease Activity ELISA is based on a universal substrate for proteases, beta casein which is labeled with biotin and coupled on beads. The cleaved fragments of biotinylated beta casein are detected with an enzyme linked immuno assay (ELISA). The combinations of bead technology and ELISA have a synergistic effect. The sensitivity and specificity via antibodies is a feature of ELISA and the beads have no scale limit and can be used in variable volumes.


Related Products

HtrA1 ELISA Assay Kit
HtrA3 ELISA Assay Kit
MMP-13 ELISA Assay Kit

Additional Information

Assay Procedure


  1. Set up the UPA microtiter plate with sufficient wells for standards and test samples as required for a double estimation. Recommended positions of Fragment-standard (0 – 2.5 nM) are microtiter plate rows 1 and 2. Recommended positions for standard HtrA1 reactions (0 – 20 nM) are plate rows 3 and 4. All other wells can be used for test sample reactions.
  2. Pipette 100 μl of each Fragment working standard into the appropriate wells.
  3. Pipette 100 μl of each Standard HtrA1 reaction into the appropriate wells.
  4. Pipette 100 μl of test sample reactions into remaining wells.
  5. Seal the plate with the provided foil and incubate on a shaker at room temperature for 120 minutes.
  6. Dilute Wash Buffer as described in page 7.
  7. Aspirate and wash the wells 4 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
  8. Blot the plate on paper towels to remove residual fluid from the plate.
  9. Add 100 μl of diluted Antibody Solution HH1 into each well.
  10. Seal the plate and incubate on a shaker at room temperature for 90 minutes.
  11. Aspirate and wash the wells 4 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
  12. Blot the plate on paper towels to remove residual fluid from the plate.
  13. Add 100 μl of diluted Antibody Solution AM into each well.
  14. Seal the plate with the provided foil and incubate on a shaker at room temperature for 30 minutes.
  15. Aspirate and wash the wells 6 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
  16. Blot the plate on paper towels to remove residual fluid from the plate.
  17. Add 100 μl of TMB Substrate to each well.
  18. Seal the plate with foil provided and incubate in the dark at room temperature for 10 minutes.
  19. Stop the reaction by adding 100 μl of Stop Solution to each well.
  20. Read the plate at 450 nm (540, 570 or 620 nm reference filter) within 30 minutes. Reading of the plate without reference may yield higher absorbencies and thus may be less accurate.

Standard Curve


Manual

Product Manual