About Protease
1. You can use Casein or Azocasein, the methods are similar, same principle: You measure the
ammount of low molecular weight molecules, released from sustrate (casein or azocasein)
that are not precipitated by TCA. Supernatant in recovered by centrifugation and then you
measure absorbance: Casein method, at 280nm using a UV cuvettes. Azocasein method, at
440 nm, in any type of cuvettes. But there is a difference about what is measured. In casein
method you measure free aminoacids (aromatic ones) that absorbe al UV region (275-280 nm,
Tyrosine and Tryptophane, and 267 Phenylalanine), and also peptides containing them. In
contrat in Azocasein method you measure any free aminoácid or peptide contaning the Azo
dye. It means that in the first one you measure aminoacids, and in the second one you
measure the dye.
Both methods are end point, well acepted, you use the better for your interest.
A tyrosine standard curve is used, in the Casein Method, to calculate activity in terms of
tyrosine equivalente released by time.
In Azocasein method, usually activity is expressed in terms of Aborsorbance increase at 440
nm by time.
2. As I understand it, azocasein is casein modified with a dye, allowing a simple continuous,
colorimetric assay for proteolysis (absorbance at 440 nm), rather than an endpoint assay.
Thus, using azocasein should allow more data to be collected in the same amount of time,
especially if one is using a plate reader and multiwell assay plates.
3. Hi Abhas, just to add to Katarzyna's answer, both casein and azocasein are acceptable assays
for measuring proteinases activities.
The only advantage that azocasein has is that its degradation products are easier to measure
by regular spectrophotometers.
If you can measure casein degradation products by UV-Vis spec, then that will be good
enough for publication.
4. both are accepted..caseinolytic you check at 660nm and Azocasein degradation at 440nm..
and you can also study action of your protease(s) on casein, azocasein and hemoglobin.. that
will add to your data and interpretations on substrate specificity while characterizing the
protein..