migrates to the longest distance on the gel. However, in the
presence of a protein/peptide which can specifically bind to this
DNA fragment, the protein binds to DNA and forms stable
protein/DNA complexes. The migration of these larger protein/
DNA complexes is retarded or shifted on the gel since they move
more slowly than the unbound DNA (Fig. 1a). There will be a
tendency to show the more protein concentration added to a fixed
concentration of DNA probe, the more shifted protein/DNA
complexes accumulate. These increasing protein/DNA complexes
will ultimately reach a plateau, once DNA is fully bound and
saturated by protein (Fig. 1b).
7. Gel retardation assay revealed that N/P ratio-dependent interactions between CPP and DNA were maximized at N/P ratios
above 12 [8].
8. Cells are grown and maintained in a 100-mm tissue culture
dish with 10 mL culture medium routinely. Cells can be seeded
in 24-well culture plates for fluorescent image studies, and the
final reaction volume is 150 μL of L5a/DNA complexes. Suspension cell culture has not been tested, but CPPs should
penetrate most cells.
9. The presence of 10% FBS in media reduces the efficiency of
complexing CPPs with the cargos leading to a decrease in the
uptake of CPPs or their complexes/conjugates [16].
10. The efficiency of the EGFP reporter gene expression in the cells
can be quantified by the flow cytometric analysis, or semiquantified by the intensity of fluorescent images [8]. As stated
above, the green fluorescent intensity can be converted and
semi-quantified using the UN-SCAN-IT or ImageJ software.
For instance, the transfection efficiency of L5a-mediated delivery
of the EGFP reporter gene expression showed approximately
41-fold increase compared to the negative control in A549
cells, while the transfection efficiency of Lipofectamine 2000mediated delivery of gene expression showed about 64-fold
increase compared to the negative control [8].
Acknowledgments
This work was supported by the Center for Biomedical Research at
Missouri University of Science and Technology, and the Ministry of
Science and Technology, Taiwan.
120
Natalie J. Holl et al.
presence of a protein/peptide which can specifically bind to this
DNA fragment, the protein binds to DNA and forms stable
protein/DNA complexes. The migration of these larger protein/
DNA complexes is retarded or shifted on the gel since they move
more slowly than the unbound DNA (Fig. 1a). There will be a
tendency to show the more protein concentration added to a fixed
concentration of DNA probe, the more shifted protein/DNA
complexes accumulate. These increasing protein/DNA complexes
will ultimately reach a plateau, once DNA is fully bound and
saturated by protein (Fig. 1b).
7. Gel retardation assay revealed that N/P ratio-dependent interactions between CPP and DNA were maximized at N/P ratios
above 12 [8].
8. Cells are grown and maintained in a 100-mm tissue culture
dish with 10 mL culture medium routinely. Cells can be seeded
in 24-well culture plates for fluorescent image studies, and the
final reaction volume is 150 μL of L5a/DNA complexes. Suspension cell culture has not been tested, but CPPs should
penetrate most cells.
9. The presence of 10% FBS in media reduces the efficiency of
complexing CPPs with the cargos leading to a decrease in the
uptake of CPPs or their complexes/conjugates [16].
10. The efficiency of the EGFP reporter gene expression in the cells
can be quantified by the flow cytometric analysis, or semiquantified by the intensity of fluorescent images [8]. As stated
above, the green fluorescent intensity can be converted and
semi-quantified using the UN-SCAN-IT or ImageJ software.
For instance, the transfection efficiency of L5a-mediated delivery
of the EGFP reporter gene expression showed approximately
41-fold increase compared to the negative control in A549
cells, while the transfection efficiency of Lipofectamine 2000mediated delivery of gene expression showed about 64-fold
increase compared to the negative control [8].
Acknowledgments
This work was supported by the Center for Biomedical Research at
Missouri University of Science and Technology, and the Ministry of
Science and Technology, Taiwan.
120
Natalie J. Holl et al.