20 µg of protein lysate from transfected COS-7 cells were fractionated on one-dimensional polyacrylamide gels and analyzed using western blotting with a goat polyclonal antibody against hERG (Santa Cruz Biotechnology, ref: SC-15968), a rabbit polyclonal antibody against GFP (Invitrogen Molecular Probes, ref: A11122), and a mouse monoclonal antibody against GAPDH (Santa Cruz Biotechnology, ref: SC-32233). Bound antibodies were detected using horseradish peroxidase-conjugated rabbit anti-goat (Santa Cruz Biotechnology, ref: SC-2922), goat anti-rabbit (Santa Cruz Biotechnology, ref: SC-2054), and goat anti-mouse (Santa Cruz Biotechnology, ref: SC-2055) secondary antibodies. Stain free gel technology (Bio-Rad) was used as loading control for protein normalization: band intensities were first normalized to the intensity of the corresponding stain free membrane lane, and ratios were then normalized to control hERG condition35 (link).
Stain free gel technology
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Stain-free gel technology is a laboratory equipment that provides a simple and efficient method for visualizing proteins and nucleic acids on polyacrylamide or agarose gels without the use of traditional staining techniques. The technology utilizes a proprietary dye that binds directly to the target molecules, eliminating the need for time-consuming staining and destaining steps.
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2 protocols using stain free gel technology
1
Quantitative Western Blot Analysis of hERG Protein
2
Molecular Characterization of hiPS-CMs and HEK-hERG
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For gene expression analysis, total RNA was extracted and reverse transcribed, from hiPS-CMs and HEK-hERG cells, as previously described [26] . PCR amplification was performed using FAM-labeled TaqMan probes: ACTB (Hs99999903_m1), KCNH2 (Hs04234270-g1) and Troponin I (Hs00165957-m1).
For Western blot analysis, 60 µg of protein lysate from hiPS-CMs or 10 µg of protein lysate from HEK-hERG, both treated for 24 h with 200 ng/ml Tat or buffer were incubated with mouse antisera against hERG protein (Santa Cruz). Secondary antibody staining was performed using goat anti-mouse IgG-HRP antibody (Santa Cruz). Stain Free gel technology (Bio-Rad) was used as loading control for protein normalization: the amount of total proteins in each lane of the blot was calculated and used for normalization [29, 30] .
For Western blot analysis, 60 µg of protein lysate from hiPS-CMs or 10 µg of protein lysate from HEK-hERG, both treated for 24 h with 200 ng/ml Tat or buffer were incubated with mouse antisera against hERG protein (Santa Cruz). Secondary antibody staining was performed using goat anti-mouse IgG-HRP antibody (Santa Cruz). Stain Free gel technology (Bio-Rad) was used as loading control for protein normalization: the amount of total proteins in each lane of the blot was calculated and used for normalization [29, 30] .
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