β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 150µl of Assay 2X Buffer to sample. Incubate the reactions at 37°C for 30 minutes or until a faint yellow color has developed	Read the absorbance at 420nm.

Protocol tips
Add 150µl of Assay 2X Buffer to sample. Incubate the reactions at 37°C for 30 minutes or until a faint yellow color has developed
Downstream tips
Read the absorbance at 420nm.

Publication protocol

Transfection. Complexes were formed as follows and added drop-wise to cell media 48 hours after seeding. All complexes were formed with either pEGFP-LUC plasmid DNA (Clonetech, Mountain View, CA), which encodes both the enhanced GFP and firefly luciferase protein (LUC), or pCMV-LacZ plasmid (Clonetech), which encodes the β-Galactosidase (β-gal) protein; both plasmids nonintegrating, produce transient transfection, and are under the direction of a CMV promoter. Plasmids were purified from Escherichia coli bacteria using Qiagen (Valencia, CA) regents and stored in Tris-EDTA buffer (10 mmol/l Tris, 1 mmol/l EDTA, PH 7.4) at −20 °C. Lipoplexes were formed with Lipofectamine LTX (LF-LTX) or Lipofectamine 2000 (LF-2000) (both Life Technologies, Grand Island, NY), following manufacturer's instructions. For LF-LTX, complexes were formed in serum-free, Opti-MEM media (Life Technologies) by first incubating plasmid DNA and “Plus” reagent diluted in media for 10 minutes at room temperature (RT), then adding transfection reagent diluted in media to the DNA and “Plus” reagent and incubating an additional 30 minutes at RT. For LF-2000, complexes were formed in serum-free, Opti-MEM media by adding transfection reagent diluted in media drop-wise to DNA in media and incubating for 20 minutes at RT. Lipoplex transfection conditions were optimized by varying lipid to DNA ratio and amount of DNA and optimized for high transfection and low cytotoxicity at 1:2 (LF-LTX) and 1:1.5 (LF-2000) lipid:DNA ratio and 0.28 µg DNA/cm2 (data not shown). For polyplexes, branched 25 kDa PEI (Sigma-Aldrich) was dissolved in Tris-buffered saline at 1 mg/ml and stored at −20 °C. Polyplexes were formed in 1× Tris-buffered saline solution by dropwise addition of PEI solution to DNA solution, briefly vortexed for 10 seconds, and incubated for 15 minutes at RT. Polyplex transfection conditions were optimized by varying nitrogen to phosphate ratio (N:P) and amount of DNA. Optimal conditions, for high transfection and low cytotoxicity at N:P of 20 and 0.32 µg DNA/cm2 of pDNA were used (data not shown).

Fluorescence microscopy. Microscopy was conducted at 24, 48, and 72 hours following DNA delivery to confirm eGFP protein expression and evaluate cell morphology using a Lecia DMI 3000B fluorescence microscope (Lecia Microsystems GmbH, Wetzlar, Germany).

Protein assays. Cells were washed with 1× PBS, then lysed with 200 µl of 1× reporter lysis buffer (Promega, Madison, WI) and gentle rocking for 15 minutes at RT. Lysates were stored at −80 °C if not analyzed immediately. Luciferase expression levels were quantified by luminescence in relative light units with a Luciferase Assay kit (Promega) and luminometer (Turner Designs, Sunnyvale, CA). β-gal activity was quantified by colorimetric assay using the β-Galactosidase Enzyme Assay System (Promega) and spectrophotometer (Beckman Coulter, Indianapolis, IN); absorbances collected at 420 nm. Both Luciferase and B-gal transfection levels were normalized to total protein levels determined with a Pierce BCA protein colorimetric assay (Pierce, Rockford, IL); absorbances collected at 562 nm.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Reporter gene assay β-galactosidase substrates - human MSCs (mesenchymal stem cells) using β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer from Promega.

Manufacturer protocol

Download the product protocol from Promega for β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Reporter gene assay β-galactosidase substrates - human MSCs (mesenchymal stem cells) using β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer from Promega. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.