Alexa fluor 647 goat anti rabbit igg a21245

Cells were lysed 48 h post-transfection using buffer containing (in mM): 150 Tris-NaCl, 25 Tris HCl, 10 Na-EGTA, 20 Na-EDTA, 5 glucose, and supplemented with 1% Triton X-100, 50 µg/ml 1,10 phenathroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine and 1x Complete Minitab (Roche Applied Science). Solution was sonicated, incubated on ice for 30 min, and centrifuged for 10 min at 13,000 rpm at 4°C. Protein concentration of supernatant was assessed using a BIORAD protein Assay. 30 µg of protein per lane was electrophoresed on a 7.5% SDS-polyacrylamide gel and then transferred onto PVDF membranes. Membranes were blocked and incubated overnight with 1:2000 anti β-actin (ab8226, abCam), and either anti-hERG (CT) (pan) (ALX-215-049-R100, Enzo Life Sciences) or anti-Ribosomal Protein L13A (C-11) (sc-390131, Santa Cruz Biotechnology). Membranes were washed and then incubated with 1:1000 dilutions of secondary antibody Alexa Fluor® 647 Goat Anti-Rabbit IgG A-21245 (Life Technologies) for 1 hour and imaged using a Chemidoc-MP Imaging System (BIORAD). Individual values were calculated from the mean of three parallel experiments completed on the same day and under identical conditions (i.e. n = 1). Protein values were then paired with experiments completed on the same day to increase statistical power.

Paraffin-embedded mouse skin sections were analyzed by immunofluorescence microscopy using fluorescence-labeled anti-mouse Alexa Fluor 568 goat anti-mouse IgG (A11031) and Alexa Fluor 647 goat anti-rabbit IgG (A21245) secondary antibodies (Life Technologies). Images were acquired on a Zeiss Axiovert 200M microscope and further processed using AxioVision software (Zeiss).