Triton X‐114 temperature‐induced phase separation method was used to isolate membrane fraction per a published protocol.3 (link) Aliquots of 100‐μg protein extracts were electrophoresed, transferred to a polyvinylidene difluoride membrane and probed with antibodies against KCNQ1 (1:1000 dilution; Merck Millipore), Na+/K+ ATPase (1:800 dilution; Proteintech), and GAPDH (1:1000 dilution; Santa Cruz).
Na k atpase
Manufactured by Proteintech
The Na+/K+ ATPase is an enzyme that plays a crucial role in maintaining the electrochemical gradient across the cell membrane. It is responsible for the active transport of sodium and potassium ions, which is essential for various cellular processes such as cell signaling, osmoregulation, and nutrient uptake.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Sodium butyrate, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ifn β, by Bioss Antibodies (1 mentions)
Nuclear factor nf κb p65, by Cell Signaling Technology (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Affinipure goat anti rabbit igg, by Proteintech (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Gapdh, by Proteintech (1 mentions)
4 protocols using na k atpase
1
Membrane Protein Isolation and Analysis
2
Transporter-Mediated Pharmaceutical Uptake Study
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SCU (purity ≥98%, HPLC) was purchased from Kunming Double Star Technology Development Co.Ltd. RSV calcium (purity ≥98%, HPLC) was purchased from Zhejiang Xindonggang Pharmaceutical Co.Ltd. Atorvastatin (internal standard) and RIF (purity ≥98%, HPLC) were sourced from the National Institute for the Control of Pharmaceutical and Biological Products. Sodium butyrate (purity ≥98%, HPLC) was provided by Sigma-Aldrich. Glycyrrhizin acid (GA) (purity ≥98%, HPLC) was purchased from Shanghai Yuanye Biotechnology Co.Ltd. Polyclonal antibodies against hOATP1B3, rOATP1B2 and Na+/K + -ATPase were sourced from Proteintech Co. Ltd. The transporter gene plasmid was provided by Shanghai Jikai Gene Co. Ltd. Human embryonic kidney 293 T (HEK293T) cell line was sourced from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, P. R. China).
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted from pancreatic, jejunal, and colonic tissues using membrane and cytoplasmic or nuclear and cytoplasmic protein extraction kits (Beyotime, Jiangsu, China) following the manufacturer's instructions. Equal amounts of protein for each sample were subjected to 5%–10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 10% nonfat milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 and incubated with primary antibodies against TLR4, MyD88, TNF receptor associated factor- (TRAF-) 6, IL-1β, TRIF, TRIF-related adaptor molecule (TRAM), IFN-β (Bioss, Beijing, China), nuclear factor- (NF-) κB p65 (Cell Signaling Technology), interferon regulatory factor- (IRF-) 3 (Santa, Dallas, TX), claudin-1, occludin, ZO-1 (Sangon, Shanghai, China), β-actin, histone H3 (Cell Signaling Technology), or Na/K ATPase (Proteintech, Rosemont, IL) at 4°C overnight. Subsequently, membranes were washed before incubation with the corresponding secondary antibodies (Zhongshan Goldenbridge, Beijing, China) for 2 h at 37°C. Finally, protein bands were visualized using an enhanced chemiluminescence detection kit (Beyotime). Na/K ATPase, β-actin, and histone H3 served as internal controls for membrane proteins, cytoplasmic proteins, and nucleoproteins, respectively.
4
Characterization of Cell Membrane Proteins
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CMs characterization were performed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) method as previously described [40 (link)]. First, protein electrophoresis was performed using a standard protocol. Then, proteins bands were visualized by staining in Coomassie blue followed by overnight destaining in water. For western blot (WB) analysis, cell membrane proteins were electrophoretically blotted onto a nitrocellulose membrane. Membranes were probed using designated antibodies, including Na+/K+-ATPase (Proteintech, #14418-1-AP), pan-cadherin (bimake, #A5614), Histone H3 (bimake, #A5737), cytochrome c (bimake, #A5184), GAPDH (Proteintech, #60004-1-Ig), and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (Proteintech, #SA00001-2). Signals were visualized by ECL reagents (Millipore, USA).
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