Irdye 800 goat anti rabbit igg

Cardiomyocytes isolated from wild type and Kcnma1^−/− mice were lysed with modified RIPA buffer (Tris-HCl 50 mM, NaCl 150 mM, EDTA-Na₂ 1 mM, EGTA-Na₄ 1 mM, Na₃VO₄ 1 mM, NaF 1 mM, Nonidet P-40 1% (v/v) Na-deoxycholate 0.5% (wt/vol), and SDS 0.1% (wt/vol), pH 7.4) containing protease inhibitor (1 tablet/50 ml; Roche) and PhosSTOP™ (1 tablet/10 ml; Roche), flash freeze in liquid nitrogen and incubated 1 h at 4 °C. Samples were centrifuged for 20 min at 10,000 × g and the supernatants were collected as lysate. Proteins (50 μg/lane) were separated on 4–20% SDS/PAGE and transferred to nitrocellulose membranes. Loading was corroborated with Ponceau S staining. Nitrocellulose membranes were blocked with Intercept® blocking buffer at room temperature for 1 h and incubated overnight with anti-BK_Ca pAb (2 μg/ml, Alomone labs, APC21) and anti-Dynamin I mAb (2 μg/ml, Abcam, EP801Y, ab52611). Membranes were washed thrice with 1x Tris-buffered Saline containing Tween-20 and incubated with 0.01 μg/ml secondary Abs (IR-dye 800 goat anti-rabbit IgG; LI-COR Biosciences; 925-68071 and IR-dye 680 goat anti-mouse; LI-COR Biosciences; 925-68070) for 60 min at room temperature. After extensive washing, membranes were visualized using BioRad ChemiDoc MP.

Protein concentrations were determined by BCA assay (Thermo Scientific) using bovine serum albumin (BSA) as the standard. SDS sample buffer was added and 5 μg protein from the sarkosyl insoluble fraction and 15 μg of protein from the low-speed supernatant was separated on 4–12% SDS-PAGE gels (Bio-Rad) and electrophoretically transferred to polyvinylidene difluoride membranes, as described previously [30 (link)]. Membranes were blocked in 0.5% casein for 1 h and incubated overnight at 4 °C submerged in 0.5% casein containing primary antibodies against total human tau (CP27; 1:500), tau phosphorylated at S396/404 (PHF-1; 1:500), or GAPDH (abcam; 1:5000). Membranes were then washed three times with TBS and incubated for 1 h with fluorophore conjugated secondary antibodies Alexa Fluor 680 anti-mouse IgG (Thermo Fisher Scientific; 1:10000) and IRDye 800 goat anti-rabbit IgG (Li-Cor Biosciences; 1:10000). Membranes were then washed three times with TBS and protein bands were detected using the multiplex Li-Cor Odyssey Infrared Imaging system (Li-Cor Biosciences).

3–20 µg protein was separated on 4–12% (w/v) SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes, as described previously [38 (link)]. Membranes were blocked in 0.5% casein for 1 h and then incubated with primary antibodies overnight at 4 °C, washed three times with TBS before incubation with fluorophore-conjugated Alexa Fluor 680 anti-mouse IgG (Thermo Fisher Scientific) or IRDye 800 goat anti-rabbit IgG (Li-Cor Biosciences, Lincoln, NE, USA) secondary antibodies and washed three times with TBS. Protein bands were detected and quantified using the multiplex Li-Cor Odyssey Infrared Imaging system (Li-Cor Biosciences).