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Biochemistry JoVE (Open Access) Citable · DOI

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

DOI: 10.3791/899-v
What you'll learn
  • ✓ Perform Sigma's standardized protease activity assay using casein substrate
  • ✓ Prepare reagents and establish standard curves for enzyme quantification
  • ✓ Measure absorbance and calculate protease specific activity from results
Protocol

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

Difficulty
beginner
Total time
~1–2 hours per assay (including incubation and absorbance measurement)
Biosafety
BSL-1

Steps

1
Understand protease activity measurement principles

Learn how proteases cleave peptide bonds and why standardized assays are necessary for comparing protease activity across samples and experiments.

▶ 01:23
2
Prepare assay reagents and materials

Gather and prepare all required reagents including casein substrate, buffers, and controls according to Sigma's protocol specifications.

▶ 01:52
3
Set up protease assay and standard curves

Establish the assay format with casein substrate, prepare serial dilutions for standard curves, and arrange reaction conditions for protease samples.

▶ 04:33
4
Measure absorbance and calculate enzyme activity

Read absorbance values using a spectrophotometer, generate standard curves, and compute protease-specific activity from experimental measurements.

▶ 08:12
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