Plasma membrane protein of the soleus muscle was extracted using a plasma membrane protein extraction kit (Catalog number K268-50, BioVision Inc., USA) and following the manufacturer’s protocols and description [11 (link)]. The BioVision kit was specifically designed to purify the plasma membrane proteins, which can be utilized in a variety of applications, such as Western blotting, 2-D gels, and enzyme analyses. Briefly, the muscle tissue (100 mg) was homogenized on ice in Homogenizing Buffer with 1 mM phenyl methanesulfonyl fluoride (PMSF) until completely lysed (30–50 times). The homogenate was centrifuged at 700×g for 10 min and then the supernatant was centrifuged at 14 000×g for 30 min at 4°C. The supernatant (the cytosol fraction) was collected and resuspended in the Upper-Phase Solution with 1 mM PMSF, then we added the Lower Phase Solution, incubated it on ice for 5 min, and centrifuged it at 1000×g for 5 min at 4°C. The upper phase was carefully collected, then centrifuged at 14 000×g for 10 min at 4°C. The resulting pellet, which contained the plasma membrane protein, was determined with BCA protein assay and was stored at −80°C for subsequent Western blotting.
Plasma membrane protein extraction kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Plasma Membrane Protein Extraction Kit is designed for the isolation of plasma membrane proteins from various cell types. It utilizes a series of centrifugation steps to fractionate cellular components and enrich for plasma membrane proteins.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ecl detection system, by Bio-Rad (3 mentions)
Na k atpase, by Abcam (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Beadbug microtube homogenizer, by Benchmark Scientific (2 mentions)
Homogeneize buffer with halt protease and phosphatase inhibitor cocktail 1x, by Thermo Fisher Scientific (2 mentions)
Thermo acclaim pepmap precolumn, by Thermo Fisher Scientific (2 mentions)
Ve cadherin, by Enzo Life Sciences (2 mentions)
Anti e cadherin, by Novus Biologicals (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Cellmask dye, by Thermo Fisher Scientific (2 mentions)
Nuclear cytosolic fractionation kit akr, by Cell Biolabs (2 mentions)
Ultimate 3000 uhplc, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Sodium deoxycholate, by Fujifilm (1 mentions)
Lysyl endopeptidase, by Fujifilm (1 mentions)
Sodium taurocholate, by Fujifilm (1 mentions)
97 protocols using plasma membrane protein extraction kit
1
Plasma Membrane Protein Extraction from Soleus Muscle
2
Cell Fractionation and Immunoblot Analysis
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Cell fractionation was performed as described previously [11 (link)]. LPS-primed cells were treated with MSU for 6 h. Lysates from LPS-primed MSU-treated cells and control cells (without LPS and MSU treatments) were obtained and separated into cytosolic and plasma membrane fractions using the Plasma Membrane Protein Extraction Kit (BioVision, Milpitas, CA, USA, Cat# K268-50) according to the manufacturer’s instructions. Each fraction was subjected to 7.5% SDS-PAGE and analyzed by IB. Na+/K+-ATPase and β-actin were used as markers of the plasma membrane and cytosolic subcellular fraction, respectively.
3
Plasma Membrane Protein Extraction
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INS-1 and human pancreatic 1.1b4 cell plasma membrane extracts were prepared using a plasma membrane protein extraction kit (Biovision). The cells were washed once in cold PBS and plasma membrane protein extraction was performed according to the manufacturer's instructions using the reagents included in the kit. The protein concentration was obtained using the Bradford protein assay. NA+K+ATPASE was used as a loading control to show the same amounts of plasma membrane protein in each lane.
4
Plasma Membrane Protein Isolation
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Cell membrane proteins were isolated from whole cells using a Plasma Membrane Protein Extraction Kit (BioVision, Mountain View, California) according to the manufacturer’s instructions as previously described (55 (link)).
5
Quantitative Bile Acid Analysis Protocol
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Vancomycin hydrochloride, polymyxin B sulfate, lithocholic acid, sodium deoxycholate, sodium taurocholate, and lysyl endopeptidase were purchased from Wako Pure Chemical Industries (Osaka, Japan). QIAamp Fast DNA Stool Mini Kit was from Qiagen (Hilden, Germany). Taq DNA polymerase containing 10 × Standard buffer (Taq) and dNTPs were obtained from BioAcademia (Osaka, Japan) and synthesised PCR primers were obtained from FASMAC (Kanagawa, Japan). Sodium taurolithocholate and sodium taurochenodeoxycholate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Taurodeoxycholic acid sodium salt was purchased from Nacalai Tesque (Kyoto, Japan). Tauro β-muricholic acid sodium salt was purchased from Steraloids (Newport, RI, USA). Taurocholic acid-d5 (TCA-d5) sodium salt was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Triglyceride Quantification Assay kit was purchased from Abcam (Cambridge, UK). Plasma Membrane Protein Extraction Kit was obtained from BioVision (Milpitas, CA, USA). Pierce BCA Protein Assay Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Sequencing-grade modified trypsin (frozen) was obtained from Promega (Madison, WI, USA). Synthesised isotope-labelled peptides were obtained from Sigma-Aldrich. Other reagents were commercially available products of reagent or analytical grade.
6
Quantifying Kv11.1 Protein Expression in HEK293 Cells
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Total homogenates were obtained from HEK293 cells using standard methods (Zhou et al., 1998 (link)). Blots were probed with primary antibodies against Kv11.1. Chemiluminescent detection was performed with substrate reagents from Pierce Biotechnology. Densitometric analysis was performed with Image for Windows software (V. Beta 4.0.2; Scion). The expression level of Kv11.1 protein was monitored using Western blotting analyses. HEK293 cells expressing KCNH2-G572S were lysed, and the total proteins (TP) were extracted. Moreover, the total protein membrane (PM) was purified using a plasma membrane protein extraction kit according to the manufacturer's instructions (Biovision, Inc. USA). TP or PM (150 μg) per sample was separated by 10% SDS-PAGE and blotted onto nitrocellulose membrane (Stratagene, La Jolla, CA). Subsequently, hERG protein was detected using primary antibodies against Actin (Affinity Reagents), the specific polyclonal rabbit anti-hERG antibody (Santa Cruz Biotechnology, CA) and goat anti-rabbit Alexa Fluor 700 (Molecular Probes, Eugene, OR, dilution 1:2000). Densitometry and the Scion Image Software (Scion, Frederick, MD) were used to quantify the band densities. All data were normalized against Actin (n = 3).
7
Plasma Membrane Protein Extraction
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Plasma membrane extracts of unsorted KCs were prepared using a plasma membrane protein extraction kit (Biovision). Cells were harvested at 5–10 × 108 in cold PBS, spun down, washed once in cold PBS and frozen. Homogenization (with Dounce homogenizer) and plasma membrane protein extraction were performed according to the manufacturer’s instructions using buffers included in the kit. Plasma membrane fraction was tested for the presence of plasma membrane markers such as Na-ATPase.
8
Cell Membrane Isolation and Analysis
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For cell membrane isolation assay, plasma membrane extracts were prepared using a plasma membrane protein extraction kit (BioVision). Extraction of plasma membrane and cytosolic proteins were performed according to the manufacturer's instructions. For cell surface protein isolation assay, cell surface proteins were biotinylated and isolated using a Pierce cell surface protein isolation kit (Thermo Scientific). Isolation of cell surface proteins was performed according to the manufacturer's instructions. Each fraction was tested for the presence of plasma membrane marker Na+/K+-ATPase and cytosolic marker GAPDH by western blotting.
9
AAT Protects Cells from Cytokine-Induced Damage
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Human islets or βTC3 cells were incubated with AAT for 2 h before the addition of cytokines (50 U/ml TNFα + 50 U/ml IL-1β + 1000 U/ml INF-γ) for human islets, and 100 U/ml IL-1β and 1000 U/ml IFN-γ for βTC3 cells. For CPZ group, islets were incubated in CPZ for 1 h before AAT pretreatment. Samples were collected and lysed using RIPA Lysis and Extraction buffer (Thermo Fisher). Cytosolic or membrane fractions were extracted with the Plasma Membrane Protein Extraction Kit (Biovision), using the manufacturer's protocol. Lysates (10-30 µg) were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies against phospho-JNK, total JNK, β-actin, ATPase Na+/K+ transporting subunit α 1 (ATP1A1), GAPDH (Cell Signaling Technology) and AAT (Sigma). Horseradish peroxidase-conjugated secondary antibodies were from Cell Signaling Technology. Signals were visualized using an ECL detection kit (Thermo Scientific). Relative protein expression of genes was quantified using ImageJ (NIH) or Image Lab™ Software (Bio-Rad).
10
Whole Cell Lysis and Membrane Fractionation
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Whole cellular lysate generation and immunoblot analysis was performed as described previously.13 (link) For membrane fractionation, cells were processed with a plasma membrane protein extraction kit (BioVision, Zuerich, Switzerland) following the manufacturer's instructions.
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