4-Methylumbelliferyl-β-D-Galactoside

Experimental protocol for β-galactosidase activity detection based on 4-Methylumbelliferyl-β-D-Galactoside

This experimental protocol is derived from the literature. Please make modifications according to your specific requirements.

1.Solution preparation

(1) Substrate solution:

2.5mg of 4-methylumbelliferyl-β-D-galactoside was weighed and dissolved in 1ml of citrated acid-sodium phosphate buffer (pH 4.5), stored in the dark at 4°C, and prepared immediately for use.

(2) 4-methylumbelliferone (4-MU) reference material Solution series:

Stock solution A (1mM): 4-MU 1.98mg dissolved in 10ml distilled water and diluted to different concentrations: 0, 0.1, 0.5, 1, 5, 10μM.

(3) Reaction buffer:

Citrate-sodium phosphate buffer (50mM, pH 4.5) containing 0.45% NaCl.

2. Sample preparation

Jurkat cells were harvested, washed twice with PBS, added cell lysate, lysed at 4°C for 30min, centrifuged at 12,000rpm for 10min, and the supernatant was removed for protein quantification.

3. Enzyme Assay:

The enzymatic reaction was initiated by mixing:

(1) 250μl lysed cell suspension with 0.1% albumin (220μl cell suspension in PBS lysed immediately after adding 30μl cell lysis buffer).

(2) 750μl 4-Methylumbelliferyl-β-D-Galactoside in 50mM citrate-phosphate buffer, pH 4.5 with 0.45% NaCl. The final substrate concentration in the reaction mixture was 0.8mM.

(3) The mixture was incubated at 37°C for 30min. After the time, the enzymatic reaction was terminated by adding 10ml 100mM glycine-NaOH buffer, pH 10.7. The same protocol was used for the blank mixture.

(4) The relative fluorescence (excitation 365nm, emission 445nm) was measured using a spectrofluorimeter. The results were compared with a 4-MU standard curve. The enzymatic activity was expressed as μU per number of cells, where U is the amount of enzyme that catalyzes the conversion of 1μM of substrate per 1min.

References:
[1] Kwapiszewski R, Szczudlowska J, Kwapiszewska K, et al. Determination of acid ?-galactosidase activity: methodology and perspectives[J]. Indian Journal of Clinical Biochemistry, 2014, 29(1): 57-62.