Chemi doc system

To evaluate the usefulness of one-tube nested real-time PCR assay, the results of conventional PCR and nested PCR were compared. PCR primers for the conventional PCR and nested PCR used in this study were selected from the nucleotide sequence of the PCMV DNA polymerase gene determined by Hamel (10 (link)) and Fryer (11 (link)), respectively. PCR was performed using 20 μL of reaction mixture (Genetbio, Daejeon, Korea) containing 2 × master mix, 1 × primer mixture, 3 μL of sample DNA, and ddH₂O added to achieve a final volume of 20 μL. The reaction conditions for conventional PCR and nested PCR using the outer primers (PCMVF1 and PCMVR1) were as follows: pre-denaturation at 94°C for 5 min; 40 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s; and a final extension at 72°C for 10 min. For nested PCR using the inner primers (PCMVFB and PCMVR2), two microliters of the first-round PCR mixture was transferred to 20 μl of a premixed solution containing the PCR reagents at the same concentrations listed above. The amplification procedure was repeated for 40 cycles with the same time and temperature parameters as described above, except that annealing at 55°C for 30 s was used. The amplified target was visualized as a single band corresponding to a length of 413-bp for conventional PCR and 160-bp for nested PCR using the Chemi Doc system (Vilber Lourmat, Deutschland, Germany).

Protein samples were quantified by Bradford assay using PRO-Measure solution (Intron, #21011) and subjected to protein electrophoresis (SDS-PAGE). Gels were blotted by wet transfer using a Bio-Rad Power Pac at 350 V for 1 or 2 h. Membranes were blocked and incubated with primary antibodies for overnight at 4 ℃. After washing, the membranes were incubated with secondary antibodies overnight at 4 °C. Next, after a washing step, the protein bands were detected using an ECL kit (XLS025-0000, Cyanagen) on the ChemiDoc system (Fusion Solo, Vilber Lourmat).
The primary antibodies used were: β-actin [Rabbit polyclonal antibody (Rab Poly Ab), Santa Cruz, sc-130656], HK1, HK2, PKM2, PDH [Rab Poly Ab, Cell Signaling Technology (CST), #8337], eIF2α (Rab Poly Ab, CST, #9722), peIF2α [Rabbit monoclonal antibody (Rab mono Ab), CST, #3597], CHOP (Mouse Monoclonal antibody, Invitrogen, #MA1-250), ANP (Rab poly Ab, Abcam, ab14348), SPT1 (Rab poly Ab, ABclonal, A6750), and PGRMC1 (Rab mono Ab, CST, #13856). The secondary antibodies used were: Mouse anti-rabbit IgG (211-032-171, Jackson ImmunoResearch, 1:5000) and Goat anti-mouse IgG antibody (BS-0296G-HRP, Bioss, 1:5000).