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InformationTRANSMEMBRANE PROTEIN EXTRACTION REAGENT (tmPER-100; Membrane Protein Isolation)
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Transmembrane Protein Extraction Reagent
(Plasma Membrane Protein Isolation. Mammalian and Plants Cells)
Part: tmper-100. 50 ml volume.
Suitable for 150, 10 cm cell culture dishes of confluent cells. Number of extractions are scalable to the cell culture dish surface area. In addition to mammalian cells, applicable to plant protein extraction - see references below.
Transmembrane Protein Extraction
The FIVEphoton Biochemicals Transmembrane Protein Extraction Reagent (tmPER-100TM) is a cell lysis-protein extraction buffer with proprietary ingredients assisting in the extraction and isolation of high molecular weight multiple-membrane spanning proteins that are otherwise poorly resolved in standard cell lysis buffers due to aggregation tendency, lipid raft association or other insolubility issues. The Transmembrane Protein Extraction Reagent is designed to extract proteins with 4 or more transmembrane domains, yet will also effectively resolve less complex transmembrane proteins. The transmembrane protein extraction reagent is applicable to transmembrane proteins that do not appreciably solubilize or resolve with RIPA buffer.
Examples for the applicability of the FIVEphoton Biochemicals Transmembrane Protein Extraction Reagent include resolution of ABC transporters, ion channels, ion exchangers and GPCRs for the downstream application of Western blotting.
Transmembrane Protein Extraction Reagent: General Protocol
The researcher first employs techniques described in the protocol manual to limit endocytosis and lysosomal targeting that may result in proteolytic cleavage of cytoplasmic domains of multi-membrane spanning proteins. In the second step, the Transmembrane Protein Extraction Reagent is added to dislodge cells from the cell culture dish and then to dissolve the cell membrane. Following membrane dissolution, brief centrifugation is used to remove cellular debris from a supernatant fraction that contains the extracted transmembrane proteins. The supernatant is added to Laemmli Sample Buffer, which is heated, but not boiled, prior to the resolution in SDS-PAGE gels. For Western blots, transfer takes place in a transfer buffer with added SDS and reduced amounts of methanol. The transfer is allowed to proceed approximately 25% longer than typically employed in Western blotting protocols.
An easy to follow protocol for cell lysis and preparation of samples for Western Blotting is included with the Transmembrane Protein Extraction Reagent, or can be downloaded in pdf format by clicking the manual tabs.
Image: Application of the Transmembrane Protein Extraction Reagent for Western blot analysis of a complex 12-membrane spanning protein (deltaF508 CFTR and wild-type CFTR). First lane: Western blot of the 150 kD variant of a mutant ABC transporter showing the isoform with only core glycosylation (deltaF508 CFTR). Second lane: Western blot of the wild-type variant of the same ABC transporter capturing both core and complex glycosylation (wild-type CFTR). The mature-fully processed wild-type isoform with complex glycosylation shown on the right has a molecular mass of 170 kD.
Product Citations
Animal Cells
Tissues Li, M. H., Suchland, K. L., & Ingram, S. L. (2016). Compensatory activation of cannabinoid CB2 receptor inhibition of GABA release in the rostral ventromedial medulla (RVM) in inflammatory pain. Journal of Neuroscience, 1310-16. Link to article.
Plant Cells
Protocol
Representative References of High Molecular Weight Transmembrane Proteins
tmPER-100TM is provided in 50 ml volume. (tmPER-200TM; 100 ml).
Supplemental Products
Shipping: Ships at ambient temperature. International delivery available.
kw. PLASMA MEMBRANE PROTEIN, TRANSMEMBRANE, PLANT PROTEIN EXTRACTION REAGENT, MAMMALIAN CELL PROTEIN EXTRACTION, MEMBRANE LYSIS, WESTERN BLOT PROTEIN EXTRACTION
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 Transmembrane Protein Extraction Reagent (Part No. tmPER-50; Plasma Protein Isolation) TmPER-50 (100 plates) $180.00 |  CALCIUM PHOSPHATE TRANSFECTION KIT | 200 10cm cell culture dishes | Part No. CP-4 CP-4 (200 plates) $385.00 | Transmembrane Protein Extraction Reagent (tmPER-200; Plasma Protein Isolation) TmPER-200 (300 plates) $260.00 |  ECL Chemiluminescence Kit | FIVEphoton Biochemicals | PCD-250, PCD-500 | High Sensitivity PCD-500 $179.95 |
 SODIUM ORTHOVANADATE, ACTIVATED, 200mM SOLN | Part ActVO-4 ActVO-4 Activated $145.00 |  - FIVEphoton Biochemicals-80x80.jpg "RIPA Buffer (RIPA-50)") RIPA Buffer (RIPA-50) RIPA-50 $82.00 |  PROTEASE INHIBITOR COCKTAIL BROAD SPECTRUM (100X; PI-1) PI-1 $115.95 |  PROTEASE INHIBITOR COCKTAIL w/ EDTA (100X, 1ml; PI-2) PI-2 $115.95 |
 CHAPS IMMUNOPRECIPITATION (IP) AND LYSIS BUFFER For Tissues and Cells | Part No. CIB-1 CIB-1 $175.00 |  Immunoprecipitation Buffer w/ Triton X-100 (TIB-1) TIB-1 $145.00 |  Tissue Protein Extraction Buffer (1X, 100 ml; Part No TEB-1) TEB-1 $125.00 |