β-Galactosidase Reporter Gene Staining Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Analyze the β-galactosidase expression 40-72 hr Add lysis buffer and incubate at room temperature for 15 minutes. Add 2x assay buffer and incubate at 37 °C for 30 minutes or until a yellow color develops (t min).	Read OD at 420 nm

Protocol tips
Analyze the β-galactosidase expression 40-72 hr Add lysis buffer and incubate at room temperature for 15 minutes. Add 2x assay buffer and incubate at 37 °C for 30 minutes or until a yellow color develops (t min).
Downstream tips
Read OD at 420 nm

Publication protocol

In vitro delivery of β-galactosidase by CPPs
Each CPP-PLP was conjugated with β-galactosidase (5 mg/mL in DMF, Sigma Aldrich) using transglutaminase (1 mg/mL, Sigma Aldrich) in conjugation buffer (50 mM Tris-HCl pH7.4, 5 mM CaCl2, 1 mM DTT) for 1 h at 37 °C. Cells were washed twice with serum-free DMEM and incubated in a mixture of serum-free medium (400 μL), the resulting conjugate solution (100 μL) at 37 °C for 6 h, washed thrice with serum-free DMEM and analyzed using flow cytometry and confocal laser scanning microscopy at 37 °C for 3 h. For microscopic analysis, cells were fixed in 1× fixation buffer (2% formaldehyde and 0.2% glutaraldehyde in PBS) for 10 min and washed twice with PBS. Using a β-galactosidase staining kit (GALS, Sigma Aldrich, St. Louis, MO, USA), cells were incubated with X-gal substrate at 37 °C for 2 h and washed twice with PBS. Stained cells were analyzed using a Nuance microscope (Nuance, Olympus; 100 × oil immersions). To quantify the activity of delivered β-galactosidase, cells containing CPP-β-galactosidase conjugates were treated as described above, except using O-nitrophenyl-β-galactoside (ONPG, Sigma Aldrich, St. Louis, MO, USA) as the substrate instead of X-gal. Cells were lysed with RIPA lysis buffer at room temperature for 10 min and centrifuged at 12,000 × g for 5 min. Supernatants were incubated with ONPG substrate (in 1 mM MgCl2, 45 mM β-mercaptoethanol, 0.1 M sodium phosphate pH 7.4) in a 96-well plate at 37 °C for 30 min before adding stop solution (3,3’,5,5’-tetramethylbenzidine, TMB; Thermo Scientific). Absorbance was measured at 405–420 nm using a spectrophotometer (Molecular Devices).

In vitro delivery of Cre-recombinase by CPPs
Cre recombinase was conjugated with CPPs as described for CPP-β-galactosidase conjugates. Recombinant Cre recombinase containing a nuclear localization sequence (NLS) was purchased from Excellgen (Rockville, MD, USA). Fibroblast cells transfected with pCALNL-DsRed were seeded into confocal bottom dishes, grown to 80% confluence, washed twice with serum-free DMEM, and 500 μL of serum-free medium was added. A 100 μL solution containing conjugation buffer, CPPs (1 μM), 25 μg Cre recombinase (5 mg/mL), and 4.8 μg transglutaminase was incubated at 37 °C for 1 h. The resulting conjugates were added to the cells and incubated at 37 °C for 6 h. Cells were washed thrice with serum-free DMEM and analyzed using flow cytometry and confocal laser scanning microscopy.

In vivo distribution of β-galactosidase by CPPs
All experiments with live animals were performed in compliance with the relevant laws and institutional guidelines of Korea Institute of Science and Technology (KIST), and institutional committees have approved the experiments. Five weeks-old BALB/c mice were injected intraperitoneally with 300 μg of CPP-β-galactosidase conjugates in 0.3 mL of PBS. After 2 h, mice were sacrificed and perfused. The main organs (brain, liver, lung, kidney, heart, and spleen) were harvested, washed with PBS, fixed in 0.25% glutaraldehyde and 1.5% formaldehyde for 2 h, and developed in 1 mg/mL X-gal staining solution at 37 °C overnight. For tissue sections, organs were cut into 15 μm sections on a cryomicrotome, fixed, and stained as above. Sections were analyzed using microscopy (Nuance, Olympus; 10 ×). For quantitative enzyme assays, the excised tissues were homogenized under liquid nitrogen and lysed with lysis buffer (250 mM Tris-HCl, pH 7.2, 0.1% Triton X-100) on ice for 30 min. The lysed tissues were centrifuged at 12,000 × g for 10 min at 4 °C, and 100 μL of supernatant from each sample with normalized total protein concentration was mixed with 100 μL of assay buffer (200 mM sodium phosphate, pH 7.3, 2 mM MgCl2, 100 mM β-mercaptoethanol, 1.33 mg/ml ONPG) and incubated at 37 °C for 30 min. Na2CO3 (1 M) was added to the reaction mixtures, and absorbance was measured at 420 nm using a microwell plate reader (Molecular Devices).

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