Elastase assay using Elastin-Congo Red assay principle i needed ?
Though i understood that Elastin is a substrate for Elastase enzyme, which is bound with the elastin congo red upon incubation with the Culture supernatant.
Spectrophotometric reading with 495 gave me a 0.01 to 0.50 difference OD value between the test and negative control (MEDIA with ECR) as well as plain LB broth.
Can you explain me the value or criteria to be considered as positive for elastase production by this method ?
Thanks in advance.
The premise of the assay is that digestion of elastin by elastase allows the Congo Red dye to be released from the dye-elastin complex. The original reference for this assay is Biochemical Journal volume 78 page 156 (1961) by none other than Fred Sanger. Another useful reference for elastase methods is Methods in Enzymology volume 19 pages 113-140 (1970).
Any absorbance value that is greater than the background value by a statistically significant amount should be considered as positive. The initial rate of absorbance increase should be directly proportional to the amount of elastase present.
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