SYBR Safe DNA gel stain into agarose gel and mix gently to
avoid air bubble formation. Pour agarose gel into an electrophoresis gel tray with a comb inserted. Remove any air bubbles
under or between teeth of the comb. Allow the agarose gel to
solidify before use.
3. A horizontal gel apparatus to run the agarose gel electrophoresis.
4. A ChemiDoc XRS+ Gel Imaging System (Bio-Rad, Hercules,
CA, USA) which is set at an excitation wavelength of 302 nm
using a trans-UV light and at an emission wavelength of
548–630 nm using the standard filter to allow green fluorescence to be captured. This imaging system contains the Quantity One 1-D analysis software v4.6.9.
2.3 Fluorescent
Image Studies
1. Medium for fluorescent image studies: Prepare RPMI 1640
medium without serum. Store at 4
C.
2. Human A549 cells or any other mammalian cell line of choice.
3. A humidified 5% CO 2 incubator set at 37
C to house human
A549 cells or mammalian cell line of choice.
4. Disposable culture plates and tissue culture dishes: 24-well
culture plates, 100-mm, and 35-mm tissue culture dishes.
5. An epifluorescent or confocal microscope to capture cellular
images.
3 Methods
3.1 Assessment of
Noncovalent L5a/DNA
Complexes
To test whether L5a peptides can interact with plasmid DNA to
form noncovalent complexes, various amounts of L5a are mixed
with DNA at different nitrogen (NH 3
+ )/phosphate (PO 4
À
) (N/P)
ratios (see Note 3) followed by separation on a 0.5% agarose gel.
The mobility of L5a/DNA complexes decreases as the amount of
L5a increases, indicating the formation of noncovalent L5a/DNA
complexes.
3.1.1 Noncovalent
Binding Between L5a
and Plasmid DNA
1. Mix L5a CPPs with the pEGFP-N1 plasmid at various N/P
ratios in Eppendorf tubes (Table 1) (see Note 4).
2. Incubate L5a and DNA mixtures in 1.5 mL Eppendorf tubes at
37
C for 1 h followed by gel retardation analysis.
3.1.2 Gel
Retardation Assay
1. Fill the electrophoresis tank with 1Â TBE gel running buffer.
2. Place the casting tray with solidified 0.5% agarose gel into the
electrophoresis tank of a horizontal gel apparatus.
3. After the formation of L5a/DNA complexes, pipette
L5a/DNA complexes pre-mixed with the gel loading buffer
(containing glycerol to a final concentration of 6%) into the
wells of the gel.
116
Natalie J. Holl et al.
avoid air bubble formation. Pour agarose gel into an electrophoresis gel tray with a comb inserted. Remove any air bubbles
under or between teeth of the comb. Allow the agarose gel to
solidify before use.
3. A horizontal gel apparatus to run the agarose gel electrophoresis.
4. A ChemiDoc XRS+ Gel Imaging System (Bio-Rad, Hercules,
CA, USA) which is set at an excitation wavelength of 302 nm
using a trans-UV light and at an emission wavelength of
548–630 nm using the standard filter to allow green fluorescence to be captured. This imaging system contains the Quantity One 1-D analysis software v4.6.9.
2.3 Fluorescent
Image Studies
1. Medium for fluorescent image studies: Prepare RPMI 1640
medium without serum. Store at 4
C.
2. Human A549 cells or any other mammalian cell line of choice.
3. A humidified 5% CO 2 incubator set at 37
C to house human
A549 cells or mammalian cell line of choice.
4. Disposable culture plates and tissue culture dishes: 24-well
culture plates, 100-mm, and 35-mm tissue culture dishes.
5. An epifluorescent or confocal microscope to capture cellular
images.
3 Methods
3.1 Assessment of
Noncovalent L5a/DNA
Complexes
To test whether L5a peptides can interact with plasmid DNA to
form noncovalent complexes, various amounts of L5a are mixed
with DNA at different nitrogen (NH 3
+ )/phosphate (PO 4
À
) (N/P)
ratios (see Note 3) followed by separation on a 0.5% agarose gel.
The mobility of L5a/DNA complexes decreases as the amount of
L5a increases, indicating the formation of noncovalent L5a/DNA
complexes.
3.1.1 Noncovalent
Binding Between L5a
and Plasmid DNA
1. Mix L5a CPPs with the pEGFP-N1 plasmid at various N/P
ratios in Eppendorf tubes (Table 1) (see Note 4).
2. Incubate L5a and DNA mixtures in 1.5 mL Eppendorf tubes at
37
C for 1 h followed by gel retardation analysis.
3.1.2 Gel
Retardation Assay
1. Fill the electrophoresis tank with 1Â TBE gel running buffer.
2. Place the casting tray with solidified 0.5% agarose gel into the
electrophoresis tank of a horizontal gel apparatus.
3. After the formation of L5a/DNA complexes, pipette
L5a/DNA complexes pre-mixed with the gel loading buffer
(containing glycerol to a final concentration of 6%) into the
wells of the gel.
116
Natalie J. Holl et al.