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Mem per plus membrane extraction kit

Manufactured by Thermo Fisher Scientific
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The Mem-PER Plus membrane extraction kit is a laboratory equipment used for the isolation and purification of membrane proteins from a variety of cell and tissue samples. The kit utilizes a detergent-based method to extract membrane proteins while preserving their native structure and function.

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Lab products found in correlation

Rac1 antibody, by Merck Group (3 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (3 mentions) Gapdh, by Cell Signaling Technology (3 mentions) E cadherin, by Cell Signaling Technology (3 mentions) Antibody for β actin, by Merck Group (2 mentions) Pull down assay kit, by Cytoskeleton (2 mentions) P rex1 antibody, by R&D Systems (2 mentions) Antisera directed against, by Santa Cruz Biotechnology (2 mentions) Rat high range insulin elisa, by ALPCO (2 mentions) P rex1sirna smartpool, by Horizon Discovery (2 mentions) Dharmafect 1, by Horizon Discovery (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Rabbit anti gapdh, by Cell Signaling Technology (1 mentions) Amplex red cholesterol assay kit, by Thermo Fisher Scientific (1 mentions) β actin antibody, by Merck Group (1 mentions) Dharmafect 1 transfection reagent, by Horizon Discovery (1 mentions)

7 protocols using mem per plus membrane extraction kit

1

Membrane Protein Extraction from 293T Cells

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293T cells at 5 × 105 were seeded and the next day transfected using Lipofectamine 3000 (Thermo Fisher Scientific) with 3 μg of E.V., wild-type mM8, or the mutant mM8 constructs we generated. At 24 h posttransfection, cells were collected and membranes were extracted using the Mem-PER Plus membrane extraction kit (Thermo Fisher Scientific) according to the manufacturer’s guidelines. The purity of the membrane fractions was verified by Western blot assays probing with rabbit anti-GAPDH (Cell Signaling Technology).
Umthong S., Lynch B., Timilsina U., Waxman B., Ivey E.B, & Stavrou S. (2021). Elucidating the Antiviral Mechanism of Different MARCH Factors. mBio, 12(2), e03264-20.
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2

Cholesterol Extraction and Quantification

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HEK 293T cells preincubated with 0.5% MβCD or vehicle were collected and homogenized in lysis buffer (Tris 50 mM, NaCl 150 mM, 1% NP-40, 0.5% Sodium dexyocholate supplemented with protease inhibitor cocktail). Following homogenization, sample lysates were centrifuged at 10000x g for 30 min at 4 °C. The supernatants were collected. For HEK 293T total membrane purification, Mem-PER Plus Membrane Extraction Kit (Thermo Fisher) was used according to the manufacturer’s instruction. Free cholesterol concentrations were determined using Amplex Red Cholesterol Assay Kit (Invitrogen). Pierce BCA Protein Assay Kit (Thermo Fisher) was used to measure concentrations of total proteins.
Yao L., Wells M., Wu X., Xu Y., Zhang L, & Xiong W. (2020). Membrane Cholesterol Dependence of Cannabinoid Modulation of Glycine Receptor. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(8), 10920-10930.
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3

Investigating P-Rex1 and RhoG Signaling

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P-Rex1 antibody was from R&D Systems (Minneapolis, MN, USA). Antisera directed against RhoG was from Santa Cruz Biotechnology (CA, USA). E-Cadherin, GAPDH and HRP-conjugated secondary antibodies were from Cell Signaling (Danvers, MA, USA). Rac1 antibody was from EMD Millipore (Burlington, MA, USA). Rat high range insulin ELISA was from ALPCO (Salem, NH, USA). Target sequence for the ON-TARGETplus Non-targeting siRNA#1 (Catalog Item- D-001810–01-20) is: UGGUUUACAUGUCGACUAA. The target sequences for the ON-TARGETplus Rat Prex1(311647) siRNA-SMARTpool (Catalog Item L-093719–02-0010 that has 4 sequences that it targets) are: GCAUGGAGCGCGACGCAUA, CAACAACAACGGCGAGUAU, UCCUGAAAGUCAACGGCAA and CCAUCAACGCCCUGGACGA. The above on-target P-Rex1siRNA SMARTpool and non-targeting control siRNA (Con-si), as well as DharmaFect1, were from Dharmacon (Lafayette, CO, USA). Antibody for β-actin and all other reagents used in the current studies were from Sigma Aldrich (St. Louis, MO, USA). Mem-PER Plus Membrane Extraction Kit was from Thermo Fisher Scientific (Waltham, MA, USA). The pull down assay kit used for the Rac1 activation was from Cytoskeleton (Denver, CO, USA).
Thamilselvan V., Gamage S., Harajli A., Chundru S.A, & Kowluru A. (2020). P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 54(6), 1218-1230.
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4

Molecular Mechanisms of CARD9 Regulation

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CARD9 antibody and THP-1 (human monocyte) cell lysate were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against ERK1/2 (total and phospho), p38 MAPK (total and phospho), E-Cadherin, GAPDH and HRP-conjugated secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA). Rac1 antibody was from EMD Millipore (Burlington, MA, USA). β-actin antibody was from Sigma Aldrich (St. Louis, MO, USA). Mem-PER Plus Membrane Extraction kit was from Thermo Fisher Scientific (Waltham, MA, USA). ON-TARGETplus Non-targeting siRNA (Con-siRNA), ON-TARGETplus Rat CARD9 siRNA-SMARTpool and DharmaFect1 transfection reagent were from Horizon Discovery (Lafayette, CO, USA). Rat specific Insulin ELISA kit was from ALPCO (Salem, NH, USA). Rac1 activation kit (pull-down assay) was obtained from Cytoskeleton (Denver, CO, USA). Mastoparan and BRD2599 (CARD9 inhibitor) were purchased from Enzo Life Sciences (Ann Arbor, MI, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.
Gamage S., Hali M, & Kowluru A. (2021). CARD9 mediates glucose-stimulated insulin secretion in pancreatic beta cells. Biochemical pharmacology, 192, 114670.
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5

Investigating P-Rex1 and RhoG Signaling

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P-Rex1 antibody was from R&D Systems (Minneapolis, MN, USA). Antisera directed against RhoG was from Santa Cruz Biotechnology (CA, USA). E-Cadherin, GAPDH and HRP-conjugated secondary antibodies were from Cell Signaling (Danvers, MA, USA). Rac1 antibody was from EMD Millipore (Burlington, MA, USA). Rat high range insulin ELISA was from ALPCO (Salem, NH, USA). Target sequence for the ON-TARGETplus Non-targeting siRNA#1 (Catalog Item- D-001810–01-20) is: UGGUUUACAUGUCGACUAA. The target sequences for the ON-TARGETplus Rat Prex1(311647) siRNA-SMARTpool (Catalog Item L-093719–02-0010 that has 4 sequences that it targets) are: GCAUGGAGCGCGACGCAUA, CAACAACAACGGCGAGUAU, UCCUGAAAGUCAACGGCAA and CCAUCAACGCCCUGGACGA. The above on-target P-Rex1siRNA SMARTpool and non-targeting control siRNA (Con-si), as well as DharmaFect1, were from Dharmacon (Lafayette, CO, USA). Antibody for β-actin and all other reagents used in the current studies were from Sigma Aldrich (St. Louis, MO, USA). Mem-PER Plus Membrane Extraction Kit was from Thermo Fisher Scientific (Waltham, MA, USA). The pull down assay kit used for the Rac1 activation was from Cytoskeleton (Denver, CO, USA).
Thamilselvan V., Gamage S., Harajli A., Chundru S.A, & Kowluru A. (2020). P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 54(6), 1218-1230.
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6

Verifying SARS-CoV-2 ORF7a Localization

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To verify that SARS-CoV-2 ORF7a mutants and the SARS-CoV-2 ORF7aTMCD4 chimera localized to the plasma membrane, we used the Mem-PER Plus membrane extraction kit (Thermo Fisher Scientific) per manufacturer’s recommendation. Membrane fraction purity was verified by western blots probing for rabbit anti-GAPDH (see Immunoblotting section).
Timilsina U., Umthong S., Ivey E.B., Waxman B, & Stavrou S. (2022). SARS-CoV-2 ORF7a potently inhibits the antiviral effect of the host factor SERINC5. Nature Communications, 13, 2935.
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7

Lysopc-Induced Arachidonic Acid Quantification

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BAECs were serum-starved for 18 h followed by the addition of lysoPC (12.5 μmol/L) for 15 min. Using Mem-PER Plus membrane extraction kit (ThermoFisher, Cat no. 89842), the membrane fraction was isolated. Briefly, BAECs were rinsed with PBS x 2 and collected by centrifugation at 1000 g for 5 min. The pellet was resuspended in the permeabilization buffer and incubated at 4 °C for 10 min. The cells were then centrifuged at 16,000 g for 15 min at 4°C. The upper cytosolic fraction was removed carefully and discarded. The pellet containing the membrane fraction was resuspended in solubilization buffer and incubated at 4°C for 30 min. The sample was then centrifuged at 16,000 g for 15 min. ArA content in the membrane fraction was assessed using an ELISA assay following the manufacturer’s protocol, and the absorbance read at 450 nm using a plate reader (SpectraMAX 190).
Nolan R.P., Langer A.M, & Wilson R. (1999). A risk assessment for exposure to grunerite asbestos (amosite) in an iron ore mine. Proceedings of the National Academy of Sciences of the United States of America, 96(7), 3412-3419.
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