The azocasein protease assay is done for the activity of protease in the culture broth or in other protein samples, if protease is present it will chew up all the proteins.

Azocasein Protease Assay Protocol

1. 200uL of 2% Azzocasein in sodium bicarbonate buffer pH 8.3
2. 200uL of 0.5% sodium bicarbonate buffer
3. 100uL of Sample to be tested

• Mix all the above and incubate the tube at 37^oC for 10mins.
• Add 0.5mL of 20% TCA to each tube.
• Vortex and incubate at room temperature for 5 mins.
• Centrifuge at 10000 RPM for 5mins/4^oC.
• Add 500uL of supernatant to 1mL of 1M NaOH
• Read the Absorbance at A₄₄₀ using a spectrophotometer.
• Check for the protease activity.

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