Cells were washed with cold PBS and harvested. Plasma membrane protein was extracted using a plasma membrane protein isolation kit (Invent Biotechnologies, Inc., Eden Prairie, MN, USA). The plasma membrane fraction was separated from the cellular components (nuclei, cytosol, and organelles) according to the manufacturer’s instructions. The protein content of plasma membrane was determined with the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA).
Plasma membrane protein isolation kit
Manufactured by Invent Biotechnologies
Sourced in United States
The Plasma Membrane Protein Isolation Kit is a laboratory product designed to isolate and extract plasma membrane proteins from cells. It provides a method for the separation and purification of these specific proteins from the cellular components.
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Lab products found in correlation
Anti bak1, by Agrisera (2 mentions)
Anti bik1, by Agrisera (2 mentions)
Anti rbohd, by Agrisera (2 mentions)
Anti mpk6, by Merck Group (2 mentions)
Anti mpk3, by Merck Group (2 mentions)
Bradford protein assay kit, by Bio-Rad (2 mentions)
Pierce cell surface protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Protein assay kit, by Thermo Fisher Scientific (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Se131, by Solarbio (1 mentions)
As12 1859, by Agrisera (1 mentions)
P5747, by Merck Group (1 mentions)
F1804, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Surepage gel, by GenScript (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Goat anti rabbit lgg hrp, by Abmart (1 mentions)
Wbkls0100, by Merck Group (1 mentions)
13 protocols using plasma membrane protein isolation kit
1
Plasma Membrane Protein Extraction
2
Biotinylation of Neuronal Surface Proteins
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A receptor biotinylation assay was performed using the Pierce cell surface protein isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) as described previously (25 (link)). Primary cultures of cortical neurons were prepared as described previously (4 (link)). The surface proteins of mouse primary cultured cortical neurons at 14 days in vitro were biotinylated with EZ-Link Sulfo-NHS-SS-biotin for 30 min at 4°C. To collect the surface proteins, cells were lysed with lysis buffer and biotinylated proteins were precipitated with NeutrAvidin agarose. The collected surface proteins were analyzed by western blotting.
Membrane protein isolation was performed using a plasma membrane protein isolation kit (Invent Biotechnologies, Plymouth, MN, USA) according to the manufacturer’s instructions. The collected membrane proteins were analyzed by western blotting.
Membrane protein isolation was performed using a plasma membrane protein isolation kit (Invent Biotechnologies, Plymouth, MN, USA) according to the manufacturer’s instructions. The collected membrane proteins were analyzed by western blotting.
3
Immunoblotting of Cell Cycle Proteins
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For immunoblotting of cell cycle-associated proteins, cells were washed twice with PBS and lysed in radioimmunoprecipitation assay buffer containing protease inhibitor cocktail (Bioprince). For immunoblotting of glucose transporter 1 (GLUT1), cells were washed twice with PBS and membrane proteins were isolated using plasma membrane protein isolation kit (Invent Biotechnologies). Protein concentrations were measured using protein assay kit (Thermo Fisher Scientific). Thirty to fifty micrograms of each sample were electrophoresed on sodium dodecylsulphate polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline/ Tween-20 (TBST) for 1 h. The membranes were incubated with primary antibodies for 1 h at room temperature or overnight at 4 °C. They were then washed three times with TBST for 5 min and incubated with IRDye secondary antibody (LI-COR Biosciences) for 1 h. The membranes were visualised using an Odyssey infrared imaging system (LI-COR Biosciences). Antibodies for cyclin D1, CDK4, p21, and p27 were purchased from Cell Signalling Technology. Antibodies for β-actin and GLUT1 were purchased from Santa Cruz Biotechnology, and Abcam, respectively.
4
Plasma Membrane Protein Isolation from HepG2 Cells
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Plasma membrane protein was extracted using a plasma membrane protein isolation kit (Invent Biotechnologies, Inc., Eden Prairie, MN, USA). Briefly, HepG2 cells were seeded in 100 mm diameter culture dishes (1 × 106 cells) and incubated for 24 h. After 24 h treatments, the cells were incubated in the absence or presence of insulin (100 nM) for 20 min. Cells were harvested and washed with PBS. The plasma membrane fraction was separated from the cellular components (nuclei, cytosol, and organelles) according to the manufacturer’s instructions. Three replicates were used.
5
Isolation of Plasma Membrane Proteins
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Cells were scraped in 1 % Triton X-100/PBS or 1 % Triton X-100 + 0.1 % SDS/PBS lysis buffers supplemented with 1 mM EDTA, 0.2 mM sodium orthovanadate and fresh protease inhibitor cocktail. Nuclei were pelleted and supernatants were used for immunoprecipitation assays.
Plasma membrane protein isolation kit (SM-005, Invent Biotechnologies Inc., MN, USA) was used to fractionate cells to obtain nuclei, cytosol, organelles and plasma membrane fractions. Three P150 mm dishes with 90 % confluent cells were used to obtain cytosolic and plasma membrane fractions as per manufacturer’s instructions. Plasma membrane protein pellet was suspended in 1 % Triton X-100 + 0.1 % SDS/PBS buffer. Cytosolic fraction was brought to a final concentration of 1 % Triton X-100 and 0.1 % SDS.
Xenograft tumor pieces were placed in 500 μL of 1 % Triton X-100 + 0.1 % SDS/PBS lysis buffer supplemented with 0.2 mM sodium orthovanadate and fresh protease inhibitor cocktail (1:100) and homogenized using polytron homogenizer for 30 s - 1 min on ice. Homogenates were spun at 14,000 rpm for 20 min at 4 °C and supernatants were used for immunoprecipitation assay.
Plasma membrane protein isolation kit (SM-005, Invent Biotechnologies Inc., MN, USA) was used to fractionate cells to obtain nuclei, cytosol, organelles and plasma membrane fractions. Three P150 mm dishes with 90 % confluent cells were used to obtain cytosolic and plasma membrane fractions as per manufacturer’s instructions. Plasma membrane protein pellet was suspended in 1 % Triton X-100 + 0.1 % SDS/PBS buffer. Cytosolic fraction was brought to a final concentration of 1 % Triton X-100 and 0.1 % SDS.
Xenograft tumor pieces were placed in 500 μL of 1 % Triton X-100 + 0.1 % SDS/PBS lysis buffer supplemented with 0.2 mM sodium orthovanadate and fresh protease inhibitor cocktail (1:100) and homogenized using polytron homogenizer for 30 s - 1 min on ice. Homogenates were spun at 14,000 rpm for 20 min at 4 °C and supernatants were used for immunoprecipitation assay.
6
Isolation and Immunoblotting of Plasma Membrane Proteins
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The indicated plant tissues were harvested, weighed, and ground in liquid nitrogen. Total proteins were extracted in protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% Glycerol, 5.0 mM DTT, 2.0 mM Na2MoO4, 2.5 mM NaF, 1.5 mM Na3VO4, 0.5% [v/v] IGEPAL CA-630 [Sigma-Aldrich], 1.0 mM PMSF, 1% [v/v] protease inhibitor cocktail and 1× PhosSTOP phosphatase inhibitor cocktail [Roche]). Plasma membrane proteins were isolated using a Plasma Membrane Protein Isolation Kit (Invent, SM-005-P) according to the manufacturer’s protocol. Protein samples were separated on 4–12% precast SurePAGE gels (GenScript) at 120 V for 1.5 h and transferred onto activated PVDF membranes at 200 mA for 2 h. Immunoblotting was performed using the following antibodies: anti-BZR1 (Youke Biotechnology, Shanghai, China, YKRP082), 1:1000; anti-BRI1 (Agrisera, AS12 1859), 1:5000; anti-SERK3/BAK1 (Agrisera, AS12 1858), 1:5000; anti-FLAG (Sigma, F1804), 1:2500; anti-phosphoserine (Sigma, P5747), 1:500; anti-phosphothreonine (Cell signaling, 9381), 1:500; anti-HA (Covance, MMS-101R), 1:2500; anti-rabbit (MBL, 458), 1:10,000; and anti-mouse (Solarbio, SE131), 1:10,000.
7
Plasma Membrane Protein Isolation
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Cell membrane was separated by using Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA). According to the protocol, the cells were collected and transferred into protein extraction filter cartridges. Then the mixture was centrifuged at 14,000× g for 30 s, and the pellet was resuspended. After centrifugation at 3000× g for 1 min, the supernatant was again transferred. After that, the mixture was centrifuged at 16,000× g for 30 min, and the pellet was saved (plasma membrane).
8
PTI Components Detection Protocol
9
Plasma Membrane Protein Isolation
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Plasma membrane proteins were separated based on the Plasma Membrane Protein Isolation Kit (SM-005, Invent) protocol. The extracted protein of cells or tissues was determined for concentration with a BCA kit (Servicebio). Equal amounts of protein were electrophoresed on 10% polyacrylamide gels and then transferred onto a polyvinylidene uoride (PVDF) membrane. After blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4 o C with primary antibodies directed against clathrin, RAB5B, NMDAR1, and β-actin (Supplementary Table 1). The membranes were incubated with secondary antibodies at room temperature for 1 h and visualized using an ECL substrate (WBKLS0100, Millipore). FlourChem software (Alpha Innotech, FlourChem) was used to analyze the ratio of the gray value of each band to the β-actin band for relative expression levels. All samples were analyzed independently and in triplicate.
10
Monitoring Immune Responses in Plants
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Four-week-old plant leaves were infiltrated with sterile water (mock) or different Pst strains at OD600=0.02, and samples were collected at 0.5, 3, 6, 8h after infiltration. Three to four leaves from different plants were collected as one sample. Protein was extracted using Plasma Membrane Protein Isolation Kit (Invent) according to the manufacturer's protocol. Concentration of the cytosolic protein was determined with Bradford protein assay kit (Bio-Rad). An equal amount of protein was loaded onto SDS-PAGE gel for western blot. Different PTI components were detected by following antibodies with indicated dilution: anti-RBOHD (Agrisera), 1:1000; anti-BAK1 (Agrisera), 1:5000; anti-BIK1 (Agrisera), 1:3000; anti-MPK3 (Sigma-Aldrich), 1:2500; anti-MPK6 (Sigma-Aldrich), 1:5000.
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