PEI Quantification By Advanced Protein Assay
ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 334 (2004) 196–198 www.elsevier.com/locate/yabio Notes & Tips
A spectrophotometric assay for the quantiWcation of polyethylenimine in DNA nanoparticles
Martin Bertschinger, Sophie Chaboche, Martin Jordan¤, Florian M. Wurm
Swiss Federal Institute of Technology Lausanne, Laboratory of Cellular Biotechnology, Lausanne VD1015, Switzerland
Received 29 April 2004
Available online 28 August 2004
Since the Wrst description of polyethylenimine (PEI)1 as a gene delivery vehicle, there has been a strong interest in its use for in vivo and in vitro transfections of mammalian cells [1]. The mechanism of gene transfer by PEI is still unclear, and little is known about the formation of PEI–
DNA nanoparticles. Such questions are diYcult to address because a rapid and sensitive assay for the quantiWcation of PEI in diVerent transfection media is not available.
The only spectrophotometric assay for the quantiWcation of PEI is based on the dark blue cuprammonium complex that is formed when copper(II) ions are added to
PEI [2,3]. This complex has a strong absorbance peak at
285 nm and a weaker one at 630 nm. Unfortunately, the absorption maximum at 285 nm is close to that of DNA
(260 nm). In addition, most cell culture media contain components that absorb at this wavelength. At 630 nm, there is much less interference from biological molecules, but the signal from the cuprammonium complex is also low. We successfully adapted a commercially available protein detection assay for the quantiWcation of PEI.
All experiments described below were performed with linear 25-kDa PEI (Polysciences, Warrington, PA, USA) that we routinely use for the transfection of mammalian cells [4], but it was possible to quantitate other PEIs
(diVerent molecular weights; branched) with this method
(data not shown). The mixing of a PEI solution with the Advanced Protein Assay (APA) reagent (Cytoskeleton, Denver, CO, USA) resulted in the formation of a
colored
BIOCHEMISTRY
Analytical Biochemistry 334 (2004) 196–198 www.elsevier.com/locate/yabio Notes & Tips
A spectrophotometric assay for the quantiWcation of polyethylenimine in DNA nanoparticles
Martin Bertschinger, Sophie Chaboche, Martin Jordan¤, Florian M. Wurm
Swiss Federal Institute of Technology Lausanne, Laboratory of Cellular Biotechnology, Lausanne VD1015, Switzerland
Received 29 April 2004
Available online 28 August 2004
Since the Wrst description of polyethylenimine (PEI)1 as a gene delivery vehicle, there has been a strong interest in its use for in vivo and in vitro transfections of mammalian cells [1]. The mechanism of gene transfer by PEI is still unclear, and little is known about the formation of PEI–
DNA nanoparticles. Such questions are diYcult to address because a rapid and sensitive assay for the quantiWcation of PEI in diVerent transfection media is not available.
The only spectrophotometric assay for the quantiWcation of PEI is based on the dark blue cuprammonium complex that is formed when copper(II) ions are added to
PEI [2,3]. This complex has a strong absorbance peak at
285 nm and a weaker one at 630 nm. Unfortunately, the absorption maximum at 285 nm is close to that of DNA
(260 nm). In addition, most cell culture media contain components that absorb at this wavelength. At 630 nm, there is much less interference from biological molecules, but the signal from the cuprammonium complex is also low. We successfully adapted a commercially available protein detection assay for the quantiWcation of PEI.
All experiments described below were performed with linear 25-kDa PEI (Polysciences, Warrington, PA, USA) that we routinely use for the transfection of mammalian cells [4], but it was possible to quantitate other PEIs
(diVerent molecular weights; branched) with this method
(data not shown). The mixing of a PEI solution with the Advanced Protein Assay (APA) reagent (Cytoskeleton, Denver, CO, USA) resulted in the formation of a
colored
References: J. Pharmacol. Biomed. Anal. 31 (2003) 143–149. Bertschinger, F. Wurm, Serum-free large scale transient transfection of CHO cells, Biotechnol. Bioeng. 87 (2004) 537–545. hydrolysis of poly(2-ethyl-2-oxazoline), J. Control Release 73 (2001) 391–399.