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 2h 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 2h or until a yellow color develops (t min).
Downstream tips
Read OD at 420 nm

Publication protocol

Each CPP-PLP was conjugated with β-galactosidase
(5mg/mL in DMF, Sigma Aldrich) using transglutaminase (1mg/mL, Sigma Aldrich) in conjugation
buffer (50mM Tris-HCl pH7.4, 5mM CaCl2, 1 mM DTT) for 1h at 37 °C. Cells were washed twice with
serum-free DMEM and incubated in a mixture of serum-free medium (400μL), the resulting conjugate
solution (100μL) at 37 °C for 6h, washed thrice with serum-free DMEM and analyzed using flow cytometry and confocal laser scanning microscopy at 37 °C for 3h. For microscopic analysis, cells were fixed
in 1× fixation buffer (2% formaldehyde and 0.2% glutaraldehyde in PBS) for 10min 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 2h 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 10min and centrifuged
at 12,000× g for 5min. Supernatants were incubated with ONPG substrate (in 1mM MgCl2, 45mM
β-mercaptoethanol, 0.1M sodium phosphate pH 7.4) in a 96-well plate at 37 °C for 30min before adding stop solution (3,3’,5,5’-tetramethylbenzidine, TMB; Thermo Scientific). Absorbance was measured at
405–420nm using a spectrophotometer (Molecular Devices).

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