Typhoon imager

To confirm protein expression from the recombinant hAd5 virus, 293 cells were infected with hAd5-ZKV at a multiplicity of infection (MOI) of 5. After 48 h of incubation at 37 °C, 5% CO2, the cells were rinsed once with PBS and harvested with a 2× NuPage buffer. Samples were heated at 95 °C for 10 min, cooled, and loaded onto NuPage 4%–12% gels. Protein was transferred to nitrocellulose membrane, blocked with non-fat milk, and probed with primary antibodies (4G2 and GeneTex anti-prM (GTX-133305) Irvine, USA) in TBST. After overnight incubation, membranes were washed 3 times with TBST and blotted with fluorescently-conjugated secondary antibodies (molecular probes). After one hour in secondary, membranes were washed three times in TBST and one time in PBS, and then the fluorescent signal was captured with an Amersham Typhoon imager.

Strains were grown in LB supplemented with 100 mg/L ampicillin with 2.5 μCi/mL ³²P_i with either 0.2% arabinose or 0.2% glucose for inducing or inhibiting conditions, respectively. Bacteria were harvested at an OD₆₀₀ ~0.8 and washed with 5mL phosphate-buffered saline. ³²P-labeled lipid A was isolated as previously described and spotted onto a silica gel TLC plate (~10,000 cpm per lane)[59 (link)]. Lipids were separated using a chloroform, pyridine, 88% formic acid, and water solvent system (50:50:16:5). TLC plates were exposed to a PhosphorImager screen and analyzed using an Amersham Typhoon Imager and software.

Protein extracts were prepared by resuspending cell pellets in a buffer containing 20 mM Tris–HCl, 250 mM NaCl and 1 mM EDTA, sonicated in a Bioruptor^® bath (pulses 30′ on/30′ off for 10 min at maximum intensity) and centrifuged at 20 000 × g for 30 min at 4°C. A 34-mer oligonucleotide containing an 8-oxoG at position 16 and labeled at the 5′ end with Cy5 was hybridized to its complementary oligonucleotide containing a cytosine opposite of the 8-oxoG:C-labeled duplex. In a standard reaction, mixture protein extracts (4 μl final volume) were added to a 10 μl reaction mixture containing 150 fmol of the 8- oxoG:C labeled duplex in 20 mM Tris–HCl, 1 mM EDTA, 200 mM NaCl, 1 mg/ml BSA and 5% glycerol. After 1 h at 37°C, NaOH (0.1N final concentration) was added, and the mixture was further incubated for 15 min at 37°C and stopped by adding 4 μl of formamide dye and heating for 5 min at 95°C. The products were resolved by denaturing 7 M urea–20% PAGE. Gels were scanned with a Typhoon imager (Amersham) and band intensities were quantified with ImageQuant^® software. The control reaction containing the oligonucleotide incubated without protein extract was used to calculate the background. The percentage of 8-oxoG cleaved is calculated, after subtraction of background, as follows: % cleavage = product/(product + substrate) × 100.