Chlorophenol red-β-D-galactopyranoside

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
1. Reagents and Materials
Substrate stock solution: CPRG, dissolved in sterile double-distilled water, prepare a 16.5 mM stock solution. Filter sterilization (0.2 μm), store at 4°C in the dark.
Detection buffer: 100 mM phosphate buffer, pH 6.8. Add 1 mM MgCl? and 50 mM 2-Mercaptoethanol (HY-Y0326) (to stabilize enzyme activity and prevent oxidation).
Enzyme/sample: Samples containing β-galactosidase (such as cell lysate, induced yeast/bacterial culture supernatant, pure enzyme, etc.).
Positive control: Known active β-galactosidase.
Negative control: Detection buffer without enzyme.
Instrument: 96-well plate, microplate reader (with 540-572 nm filter), constant temperature incubator.
2. Experimental Procedures
2.1 Preparation of Working Solution: Add the CPRG stock solution to the detection buffer, and prepare a working solution with a final concentration of 2.5 mM CPRG.
2.2 Sample Addition: In a 96-well plate, add 50 μL of enzyme/sample solution and 50 μL of CPRG working solution to each well. (The total volume is 100 μL. Set duplicate wells and include positive control wells and negative control wells.)
2.3 Incubation and Reading: Immediately place the microplate in a 37°C constant temperature incubator. Start the kinetic reading program. Read the absorbance every 1-2 minutes at a wavelength of 572 nm for 30-60 minutes.
2.4 Data Analysis: Plot a reaction curve with time on the x-axis and absorbance (A572) on the y-axis. Usually, the linear growth period is selected to calculate the rate of change in absorbance over time (ΔA/min). This rate is proportional to the activity of β-galactosidase in the sample.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.