Pro-prep solution (200 μl) (Intron Biotechnology, Seongnam, Korea) was added to a pellet-shaped cell and vortexed to create a homogenous suspension. Each sample was kept on ice for 30 minutes and centrifuged at 13,000 rpm for 20 minutes. The supernatant was transferred to a new tube. The extracted proteins were quantitated using the Bradford assay, and the defined quantity of total protein was used for a western blot assay. SDS-PAGE (10%) was performed at 0.02 A for 2.5 hours. The resolved proteins were transferred to a PVDF membrane at 100 V for 1 hour. After transfer, proteins were confirmed by the Ponceau S stain and blocked with 5% skim milk for 1 hour at room temperature. Primary antibodies were against Kir4.1, NKCC1 (ab59791; Abcam, Cambridge, UK), KCNQ4 (ab65797, Abcam, Cambridge, UK), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Each primary antibody was diluted 1 : 1,000 in 2.5% skim milk and incubated overnight at 4°C. The secondary antibody was diluted 1 : 10,000 in 1% skim milk and incubated for 1 hour at room temperature. After washing five times with 1x Tris-buffered saline Tween 20 (TBST) for 5 minutes, ECL solution (Millipore, USA) was added for 1 minute, and the band was confirmed with the ChemiDoc gel imaging system (Bio-Rad, Hercules, CA, USA).
Ab65797
Manufactured by Abcam
Sourced in United Kingdom
Ab65797 is a primary antibody product. It is designed for use in immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab59791, by Abcam (1 mentions)
Chemidoc gel imaging system, by Bio-Rad (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Pro prep solution, by iNtRON Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ab17052, by Abcam (1 mentions)
Ab23905, by Abcam (1 mentions)
Lsm710 upright, by Zeiss (1 mentions)
Duolink in situ pla detection kit 563, by Olink (1 mentions)
A11126, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
3 protocols using ab65797
1
Western Blot Analysis of Ion Channels
2
Colocalization of Kv7.4, Caveolin-1, and Dynein
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Colocalization of dynein with Kv7.4 or caveolin-1 and caveolin-1 with Kv7.4 was studied with PLA in HEK293B cells stably expressing Kv7.4 and Kv7.4-Q580A or freshly isolated rat mesenteric artery myocytes using the Duolink in situ (PLA) detection kit 563 (Olink) per the manufacturer’s instructions. Similar to previous studies (Zhong et al., 2010a (link); Brueggemann et al., 2014 (link); Chadha et al., 2014 (link); Jepps et al., 2015 (link); Stott et al., 2016 (link); Barrese et al., 2020 (link)), cells were allowed to adhere to coverslips and fixed in 4% paraformaldehyde in PBS. Cells were permeabilized in PBST, blocked in Duolink blocking solution, and incubated with pairs of primary antibodies. The primary antibodies employed were dynein (ab23905; Abcam), Kv7.4 (ab65797; Abcam), caveolin-1 (1:500 ab17052; Abcam), caveolin-1 (C3237; Sigma-Aldrich), and NCX (R3F1; Swant). Combinations of secondary anti-rabbit or anti-mouse antibodies of PLA PLUS and MINUS probes were used followed by hybridization, ligation, and amplification steps. Colocalization signals (proteins located within 40 nm of each other) were visualized with a standard Zeiss LSM710 upright laser-scanning confocal microscope.
3
Localization and Quantification of Kv7.4 in Rat Mesenteric Artery Myocytes
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Freshly dissociated rat mesenteric artery myocytes were isolated as described previously by Jepps et al 16 and allowed to adhere to coverslips before being fixed in 4% paraformaldehyde (Sigma) in PBS for 30 minutes at room temperature. Cells were blocked, permeabilized, and stained as described previously. 16 Primary antibodies used were Kv7.4 (1:200; ab65797; Abcam, United Kingdom) and α-tubulin (1:1000; A11126; Invitrogen, Denmark), as well as wheat germ agglutinin Alexa Fluor 555 (as per the manufacturer's instructions; Life Technologies). Cells were visualized with structured illumination microscopy using a Zeiss Elyra PS.1 Super Resolution Microscope (Carl Zeiss, Germany). Midcell xy sections were selected and analyzed, and 3-dimensional reconstructions were created using Zen software (Carl Zeiss). Total cell fluorescence and intracellular fluorescence signals were quantified using Zen 2012 confocal software. Signals originating from the surface membrane, demarcated by wheat germ agglutinin, were obtained by subtracting the total intracellular signal from the total cell fluorescence signal, with both normalized to their respective areas, and background fluorescence was subtracted. The surface-associated signal was expressed subsequently as a percentage of the total cell fluorescence, as described previously. 17
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