What does the beta galactosidase assay measure?

β-Galactosidase converts the colorless ONPG substrate into galactose and the chromophore o-nitrophenol, yielding a bright yellow solution. The β-galactosidase activity of the solution can be quantitated using a spectrophotometer or a microplate reader to determine the amount of substrate converted at 420 nm.

How is beta galactosidase activity measured?

Beta-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-beta-D-galactopyranoside (ONPG).

How many kDa is beta galactosidase?

465.4 kDa
Additional sequence information Beta Galactosidase is a homotetramer with a molecular weight of 465.4 kDa (116.3 kDa subunits each).

How many subunits does beta galactosidase have?

Defects in the latter enzyme cause GM1 gangliosidosis. Galactocerebroside β-galactosidase can be isolated as an 80 kDa polypeptide consisting of 50 and 30 kDa subunits. The pH optimum of the enzyme is acidic. The enzyme is active against galactosylceramides that vary in fatty acid chain length and α-hydroxylation.

How to calculate Miller units for beta galactosidase?

Miller Units = 1000 x [(OD 420 – 1.75 x OD 550)] / (T x V x OD 600) OD 420 and OD 550 are read from the reaction mixture. OD 600 reflects cell density in the washed cell suspension. T = time of the reaction in minutes. V = volume of culture used in the assay in mLs.

What is the protocol for the beta galactosidase assay?

In short, the protocol consists of measuring the cell density of a culture of bacteria (Abs 600 ), then removing an aliquot of the cells from the cuvette and mixing them with a “permeabilization” solution that contains detergent which disrupts the cell membranes (but leaves the β-Gal intact). This kills the cells and stops translation.

How much Z buffer to use for beta galactosidase?

Dilute cells in Z buffer to 1 mL (most easily donewith a pippeter). For most activities, 0.5 mL cells + 0.5 mL Z bufferwill produce a desirable amount of yellow color in 1-2 hours. Forhigher levels (>500 Miller units), try 0.1 mL cells +0.9 mL Z buffer.

How to make beta galactosidase in a microcentrifuge?

1. Wash each 60-mm plate of cells twice with 5 ml of PBS buffer. 2. Add 1 ml of PBS buffer to each 60-mm plate. 3. Gently scrape down the cells with a Teflon®spatula and transfer the suspension into a 1.5-ml microcentrifuge tube. 4. Rinse the plate with 300 μl of PBS buffer and add the suspension to the microcentrifuge tube. 5.